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Abstract:

The present application relates to anti-PD-L1 antibodies, which have
therapeutic use to enhance T-cell function to upregulate cell-mediated
immune responses and for the treatment of T cell dysfunctional disorders,
including infection (e.g., acute and chronic) and tumor immunity.

4. The antibody of claim 1, wherein the anti-PD-L1 antibody further
comprises a VL and a VH framework region derived from a human consensus
sequence.

5. The antibody of claim 4, wherein the VH sequence is derived from a
Kabat subgroup I, II, or III sequence.

6. The antibody of claim 5, wherein the VH sequence is derived from Kabat
subgroup III.

7. The antibody of claim 6, further wherein the heavy chain framework
sequences are juxtaposed between the HVRs according to the formula:
(HC--FR1)--(HVR-H1)--(HC--FR2)--(HVR-H2)--(HC--FR3)--(HVR-H3)--(HC--FR4).

9. The antibody of claim 4, wherein the VL sequence is derived from a
Kabat kappa I, II, III or IV subgroup sequence.

10. The antibody of claim 9, wherein the VL sequence is derived from
Kabat kappa I.

11. The antibody of claim 9, further wherein the variable light chain
framework sequences are juxtaposed between the HVRs according to the
formula: (LC-FR1)--(HVR-L1)-(LC-FR2)--(HVR-L2)-(LC-FR3)--(HVR-L3)-(LC-FR4-
).

further wherein, the light chain comprises an HVR-L1, HVR-L2 and HVR-L3,
in which: (A) the HVR-L1 sequence is
RASQX4X5X6TX7X8A (SEQ ID NOs:8); (B) the HVR-L2
sequence is SASX9LX10S (SEQ ID NOs:9); (C) the HVR-L3 sequence
is QQX11X12X13X14PX15T (SEQ ID NOs:10); wherein:
X1 is D or G; X2 is S or L; X3 is T or S; X4 may be D
or V; X5 may be V or I; X6 may be S or N; X7 may be A or
F; X8 may be V or L; X9 may be F or T; X10 may be Y or A;
X11 may be Y, G, F, or S; X12 may be L, Y, F or W; X13 may
be Y, N, A, T, G, F or I; X14 may be H, V, P, T or I; X15 may
be A, W, R, P or T.

38. A composition comprising the antibody of claims 1-18 and a
pharmaceutically acceptable carrier.

39. A composition comprising the antibody of claims 19-37 and a
pharmaceutically acceptable carrier.

40. An article of manufacture comprising the composition of claim 39 and
at least one vaccine.

41. An article of manufacture comprising the composition of claim 39 and
at least one anti-viral agent.

42. An article of manufacture comprising the composition of claim 39 and
at least one chemotherapeutic agent.

43. The article of claim 42, wherein the chemotherapeutic agent is an
anti-VEGF antibody.

44. The article of claim 43, wherein the chemotherapeutic agent is
oxaliplatin.

45. The article of claim 43, wherein the chemotherapeutic agent is a RAF
inhibitor.

Description:

RELATED APPLICATIONS

[0001] This application is a continuation of U.S. Ser. No. 12/633,339,
filed 8 Dec. 2009, now allowed, which claims the benefit of priority
under 35 USC 119(e) of U.S. Provisional Application No. 61/121,092, filed
9 Dec. 2008, the disclosures of which are incorporated herein by
reference in their entirety.

FIELD OF THE INVENTION

[0002] This invention relates generally to immune function and to
enhancing T-cell function, including the upregulation of cell-mediated
immune responses and to the treatment of T cell dysfunctional disorders.

BACKGROUND OF THE INVENTION

[0003] Co-stimulation or the provision of two distinct signals to T-cells
is a widely accepted model of lymphocyte activation of resting T
lymphocytes by antigen-presenting cells (APCs). Lafferty et al., Aust. J.
Exp. Biol. Med. Sci. 53: 27-42 (1975). This model further provides for
the discrimination of self from non-self and immune tolerance. Bretscher
et al., Science 169: 1042-1049 (1970); Bretscher, P. A., P.N.A.S. USA 96:
185-190 (1999); Jenkins et al., J. Exp. Med. 165: 302-319 (1987). The
primary signal, or antigen specific signal, is transduced through the
T-cell receptor (TCR) following recognition of foreign antigen peptide
presented in the context of the major histocompatibility-complex (MHC).
The second or co-stimulatory signal is delivered to T-cells by
co-stimulatory molecules expressed on antigen-presenting cells (APCs),
and induce T-cells to promote clonal expansion, cytokine secretion and
effector function. Lenschow et al., Ann. Rev. Immunol. 14:233 (1996). In
the absence of co-stimulation, T-cells can become refractory to antigen
stimulation, do not mount an effective immune response, and further may
result in exhaustion or tolerance to foreign antigens.

[0004] The simple two-signal model can be an oversimplification because
the strength of the TCR signal actually has a quantitative influence on
T-cell activation and differentiation. Viola et al., Science 273: 104-106
(1996); Sloan-Lancaster, Nature 363: 156-159 (1993). Moreover, T-cell
activation can occur even in the absence of co-stimulatory signal if the
TCR signal strength is high. More importantly, T-cells receive both
positive and negative secondary co-stimulatory signals. The regulation of
such positive and negative signals is critical to maximize the host's
protective immune responses, while maintaining immune tolerance and
preventing autoimmunity. Negative secondary signals seem necessary for
induction of T-cell tolerance, while positive signals promote T-cell
activation. While the simple two-signal model still provides a valid
explanation for naive lymphocytes, a host's immune response is a dynamic
process, and co-stimulatory signals can also be provided to
antigen-exposed T-cells.

[0005] The mechanism of co-stimulation is of therapeutic interest because
the manipulation of co-stimulatory signals has shown to provide a means
to either enhance or terminate cell-based immune response. Recently, it
has been discovered that T cell dysfunction or anergy occurs concurrently
with an induced and sustained expression of the inhibitory receptor,
programmed death 1 polypeptide (PD-1). As a result, therapeutic targeting
PD-1 and other molecules which signal through interactions with PD-1,
such as programmed death ligand 1 (PD-L1) and programmed death ligand 2
(PD-L2) are an area of intense interest. The inhibition of PD-L1
signaling has been proposed as a means to enhance T cell immunity for the
treatment of cancer (e.g., tumor immunity) and infection, including both
acute and chronic (e.g., persistent) infection. However, as an optimal
therapeutic directed to a target in this pathway has yet to be
commercialized, a significant unmet medical need exists.

SUMMARY OF THE INVENTION

[0006] The present invention provides for anti-PD-L1 antibodies, including
nucleic acid encoding and compositions containing such antibodies, and
for their use to enhance T-cell function to upregulate cell-mediated
immune responses and for the treatment of T cell dysfunctional disorders,
including infection (e.g., acute and chronic) and tumor immunity.

[0007] In one embodiment, the invention provides for an isolated heavy
chain variable region polypeptide comprising an HVR-H1, HVR-H2 and HVR-H3
sequence, wherein:

In one specific aspect, X1 is D; X2 is S and X3 is T. In
another aspect, the polypeptide further comprises variable region heavy
chain framework sequences juxtaposed between the HVRs according to the
formula: (HC--FR1)-(HVR-H1)-(HC--FR2)-(HVR-H2)-(HC--FR3)-(HVR-H3)-(HC--FR-
4). In yet another aspect, the framework sequences are derived from human
consensus framework sequences. In a further aspect, the framework
sequences are VH subgroup III consensus framework. In a still further
aspect, at least one of the framework sequences is the following:

[0009] further wherein: X4 is D or V; X5 is V or I; X6
is S or N; X7 is A or F; X8 is V or L; X9 is F or T;
X10 is Y or A; X11 is Y, G, F, or S; X12 is L, Y, F or W;
X13 is Y, N, A, T, G, F or I; X14 is H, V, P, T or I; X15
is A, W, R, P or T. In a still further aspect, X4 is D; X5 is
V; X6 is S; X7 is A; X8 is V; X9 is F; X10 is Y;
X11 is Y; X12 is L; X13 is Y; X14 is H; X15 is
A. In a still further aspect, the light chain further comprises variable
region light chain framework sequences juxtaposed between the HVRs
according to the formula:
(LC-FR1)--(HVR-L1)-(LC-FR2)--(HVR-L2)-(LC-FR3)--(HVR-L3)-(LC-FR4). In a
still further aspect, the framework sequences are derived from human
consensus framework sequences. In a still further aspect, the framework
sequences are VL kappa I consensus framework. In a still further aspect,
at least one of the framework sequence is the following:

[0013] Further wherein: X1 is D or G; X2 is S or L; X3
is T or S; X4 is D or V; X5 is V or I; X6 is S or N;
X7 is A or F; Xs is V or L; X9 is F or T; X10 is Y or
A; X11 is Y, G, F, or S; X12 is L, Y, F or W; X13 is Y, N,
A, T, G, F or I; X14 is H, V, P, T or I; X15 is A, W, R, P or
T. In a specific aspect, X1 is D; X2 is S and X3 is T. In
another aspect, X4 is D; X5 is V; X6 is S; X7 is A;
Xs is V; X9 is F; X10 is Y; X11 is Y; X12 is L;
X13 is Y; X14 is H; X15 is A. In yet another aspect,
X1 is D; X2 is S and X3 is T, X4 is D; X5 is V;
X6 is S; X7 is A; X8 is V; X9 is F; X10 is Y;
X11 is Y; X12 is L; X13 is Y; X14 is H and X15
is A. In a further aspect, the heavy chain variable region comprises one
or more framework sequences juxtaposed between the HVRs as:
(HC--FR1)--(HVR-H1)--(HC--FR2)--(HVR-H2)--(HC--FR3)--(HVR-H3)--(HC--FR4),
and the light chain variable regions comprises one or more framework
sequences juxtaposed between the HVRs as:
(LC-FR1)--(HVR-L1)-(LC-FR2)--(HVR-L2)-(LC-FR3)--(HVR-L3)-(LC-FR4). In a
still further aspect, the framework sequences are derived from human
consensus framework sequences. In a still further aspect, the heavy chain
framework sequences are derived from a Kabat subgroup I, II, or III
sequence. In a still further aspect, the heavy chain framework sequence
is a VH subgroup III consensus framework. In a still further aspect, one
or more of the heavy chain framework sequences is the following:

In a still further aspect, the light chain framework sequences are
derived from a Kabat kappa I, II, II or IV subgroup sequence. In a still
further aspect, the light chain framework sequences are VL kappa I
consensus framework. In a still further aspect, one or more of the light
chain framework sequences is the following:

In a still further specific aspect, the antibody further comprises a
human or murine constant region. In a still further aspect, the human
constant region is selected from the group consisting of IgG1, IgG2,
IgG2, IgG3, IgG4. In a still further specific aspect, the human constant
region is IgG1. In a still further aspect, the murine constant region is
selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In a
still further aspect, the murine constant region is IgG2A. In a still
further specific aspect, the antibody has reduced or minimal effector
function. In a still further specific aspect the minimal effector
function results from an "effector-less Fc mutation" or aglycosylation.
In still a further embodiment, the effector-less Fc mutation is an N297A
or D265A/N297A substitution in the constant region.

[0014] In yet another embodiment, the invention provides for an anti-PD-L1
antibody comprising a heavy chain and a light chain variable region
sequence, wherein:

[0016] (b) the light chain
further comprises an HVR-L1, HVR-L2 and an HVR-L3 sequence having at
least 85% sequence identity to RASQDVSTAVA (SEQ ID NO:17), SASFLYS (SEQ
ID NO:18) and QQYLYHPAT (SEQ ID NO:19), respectively. In a specific
aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or 100%. In another aspect, the heavy chain
variable region comprises one or more framework sequences juxtaposed
between the HVRs as:
(HC--FR1)--(HVR-H1)--(HC--FR2)--(HVR-H2)--(HC--FR3)--(HVR-H3)--(HC--FR4),
and the light chain variable regions comprises one or more framework
sequences juxtaposed between the HVRs as:
(LC-FR1)--(HVR-L1)-(LC-FR2)--(HVR-L2)-(LC-FR3)--(HVR-L3)-(LC-FR4). In yet
another aspect, the framework sequences are derived from human consensus
framework sequences. In a still further aspect, the heavy chain framework
sequences are derived from a Kabat subgroup I, II, or III sequence. In a
still further aspect, the heavy chain framework sequence is a VH subgroup
III consensus framework. In a still further aspect, one or more of the
heavy chain framework sequences is the following:

In a still further aspect, the light chain framework sequences are
derived from a Kabat kappa I, II, II or IV subgroup sequence. In a still
further aspect, the light chain framework sequences are VL kappa I
consensus framework. In a still further aspect, one or more of the light
chain framework sequences is the following:

In a still further specific aspect, the antibody further comprises a
human or murine constant region. In a still further aspect, the human
constant region is selected from the group consisting of IgG1, IgG2,
IgG2, IgG3, IgG4. In a still further specific aspect, the human constant
region is IgG1. In a still further aspect, the murine constant region is
selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In a
still further aspect, the murine constant region if IgG2A. In a still
further specific aspect, the antibody has reduced or minimal effector
function. In a still further specific aspect the minimal effector
function results from an "effector-less Fc mutation" or aglycosylation.
In still a further embodiment, the effector-less Fc mutation is an N297A
or D265A/N297A substitution in the constant region.

[0017] In a still further embodiment, the invention provides for an
isolated anti-PD-L1 antibody comprising a heavy chain and a light chain
variable region sequence, wherein:

[0020] In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In another
aspect, the heavy chain variable region comprises one or more framework
sequences juxtaposed between the HVRs as:
(HC--FR1)--(HVR-H1)--(HC--FR2)--(HVR-H2)--(HC--FR3)--(HVR-H3)--(HC--FR4),
and the light chain variable regions comprises one or more framework
sequences juxtaposed between the HVRs as:
(LC-FR1)--(HVR-L1)-(LC-FR2)--(HVR-L2)-(LC-FR3)--(HVR-L3)-(LC-FR4). In yet
another aspect, the framework sequences are derived from human consensus
framework sequences. In a further aspect, the heavy chain framework
sequences are derived from a Kabat subgroup I, II, or III sequence. In a
still further aspect, the heavy chain framework sequence is a VH subgroup
III consensus framework. In a still further aspect, one or more of the
heavy chain framework sequences is the following:

In a still further aspect, the light chain framework sequences are
derived from a Kabat kappa I, II, II or IV subgroup sequence. In a still
further aspect, the light chain framework sequences are VL kappa I
consensus framework. In a still further aspect, one or more of the light
chain framework sequences is the following:

In a still further specific aspect, the antibody further comprises a
human or murine constant region. In a still further aspect, the human
constant region is selected from the group consisting of IgG1, IgG2,
IgG2, IgG3, IgG4. In a still further specific aspect, the human constant
region is IgG1. In a still further aspect, the murine constant region is
selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In a
still further aspect, the murine constant region if IgG2A. In a still
further specific aspect, the antibody has reduced or minimal effector
function. In a still further specific aspect, the minimal effector
function results from production in prokaryotic cells. In a still further
specific aspect the minimal effector function results from an
"effector-less Fc mutation" or aglycosylation. In still a further
embodiment, the effector-less Fc mutation is an N297A or D265A/N297A
substitution in the constant region.

[0021] In a still further embodiment, the invention provides for
compositions comprising any of the above described anti-PD-L1 antibodies
in combination with at least one pharmaceutically-acceptable carrier.

[0022] In a still further embodiment, the invention provides for isolated
nucleic acid encoding a light chain or a heavy chain variable region
sequence of an anti-PD-L1 antibody, wherein:

[0025] In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In aspect, the
heavy chain variable region comprises one or more framework sequences
juxtaposed between the HVRs as:
(HC--FR1)--(HVR-H1)--(HC--FR2)--(HVR-H2)--(HC--FR3)--(HVR-H3)--(HC--FR4),
and the light chain variable regions comprises one or more framework
sequences juxtaposed between the HVRs as:
(LC-FR1)--(HVR-L1)-(LC-FR2)--(HVR-L2)-(LC-FR3)--(HVR-L3)-(LC-FR4). In yet
another aspect, the framework sequences are derived from human consensus
framework sequences. In a further aspect, the heavy chain framework
sequences are derived from a Kabat subgroup I, II, or III sequence. In a
still further aspect, the heavy chain framework sequence is a VH subgroup
III consensus framework. In a still further aspect, one or more of the
heavy chain framework sequences is the following:

In a still further aspect, the light chain framework sequences are
derived from a Kabat kappa I, II, II or IV subgroup sequence. In a still
further aspect, the light chain framework sequences are VL kappa I
consensus framework. In a still further aspect, one or more of the light
chain framework sequences is the following:

In a still further specific aspect, the antibody further comprises a
human or murine constant region. In a still further aspect, the human
constant region is selected from the group consisting of IgG1, IgG2,
IgG2, IgG3, IgG4. In a still further specific aspect, the human constant
region is IgG1. In a still further aspect, the murine constant region is
selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In a
still further aspect, the murine constant region if IgG2A. In a still
further specific aspect, the antibody has reduced or minimal effector
function. In a still further specific aspect, the minimal effector
function results from production in prokaryotic cells. In a still further
specific aspect the minimal effector function results from an
"effector-less Fc mutation" or aglycosylation. In still a further aspect,
the effector-less Fc mutation is an N297A or D265A/N297A substitution in
the constant region.

[0026] In a still further aspect, the nucleic acid further comprises a
vector suitable for expression of the nucleic acid encoding any of the
previously described anti-PD-L1 antibodies. In a still further specific
aspect, the vector further comprises a host cell suitable for expression
of the nucleic acid. In a still further specific aspect, the host cell is
a eukaryotic cell or a prokaryotic cell. In a still further specific
aspect, the eukaryotic cell is a mammalian cell, such as Chinese Hamster
Ovary (CHO).

[0027] In a still further embodiment, the invention provides for a process
of making an anti-PD-L1 antibody or antigen binding fragment thereof,
comprising culturing a host cell containing nucleic acid encoding any of
the previously described anti-PD-L1 antibodies or antigen-binding
fragment in a form suitable for expression, under conditions suitable to
produce such antibody or fragment, and recovering the antibody or
fragment.

[0028] In a still further embodiment, the invention provides for a
composition comprising an anti-PD-L1 antibody or antigen binding fragment
thereof as provided herein and at least one pharmaceutically acceptable
carrier.

[0029] In a still further embodiment, the invention provides an article of
manufacture comprising a container enclosing a therapeutically effective
amount of a composition disclosed herein and a package insert indicating
use for the treatment of a T-cell dysfunctional disorder.

[0030] In a still further embodiment, the invention provides for an
article of manufacture comprising any of the above described anti-PD-L1
compositions in combination with at least one BNCA molecules. In one
aspect, the BNCA molecules is an antibody, antigen binding antibody
fragment, BNCA oligopeptide, BNCA RNAi or BNCA small molecule. In another
aspect, the B7 negative costimulatory molecule is selected from the group
consisting of: CTLA-4, PD-1, PD-L1, PD-L2, B7.1, B7-H3 and B7-H4.

[0031] In a still further embodiment, the article of manufacture comprises
any of the above described anti-PD-L1 compositions in combination with a
chemotherapeutic agent. In one aspect, the chemotherapeutic agent is
gemcitabine.

[0032] In a still further embodiment, the invention provides for an
article of manufacture comprising any of the above described anti-PD-L1
antibodies in combination with one or more agonists of a positive
costimulatory molecule. In one aspect, a positive costimulatory molecule
is a B7 family costimulatory molecule. In another aspect the positive
costimulatory molecule is selected from the group consisting of: CD28,
CD80, CD86, ICOS/ICOSL. In yet another aspect, the positive costimulatory
molecule is a TNFR family costimulatory molecule. In a further aspect,
the TNFR costimulatory molecule is selected form the group consisting of:
OX40/OX40L, 4-1BB/4-1BBL, CD27/CD27L, CD30/CD30L and HVEM/LIGHT, and
soluble fragments, constructs and agonist antibodies thereof.

[0033] In a still further embodiment, the invention provides for an
article of manufacture comprising any of the above described anti-PD-L1
antibodies in combination with one or more antibiotics. In one aspect,
the antibiotic is selected from the group consisting of an anti-viral
agent, anti-bacterial agent, anti-fungal agent, anti-protozoan agent.

[0034] In another aspect the anti-viral agent is selected from the group
consisting of reverse transcriptase inhibitors, protease inhibitors,
integrase inhibitors, entry or fusion inhibitors, maturation inhibitors,
viral release inhibitors, immune response enhancers, anti-viral
synergistic enhancers, vaccines, hepatic agonists and herbal therapies.
In yet another aspect, the combination comprises one or more categories
of anti-viral agents.

[0035] In a still further embodiment, the invention provides for an
article of manufacture comprising any of the above described anti-PD-L1
antibodies in combination with one or more vaccines.

[0036] In a still further embodiment, the invention provides for a method
of enhancing T-cell function comprising administering an effective amount
of any of the above described anti-PD-L1 antibodies or compositions. In
one aspect, the anti-PD-L1 antibody or composition renders dysfunctional
T-cells non-dysfunctional.

[0037] In a still further embodiment, the invention provides for a method
of treating a T-cell dysfunctional disorder comprising administering a
therapeutically effective amount of any of the above described anti-PD-L1
antibodies or compositions. In one specific aspect, the T-cell
dysfunctional disorder is infection or tumor immunity. In another aspect
the infection is acute or chronic. In another aspect, the chronic
infection is persistent, latent or slow. In yet another aspect, the
chronic infection results from a pathogen selected from the group
consisting of bacteria, virus, fungi and protozoan. In a further aspect,
the pathogen level in the host is reduced. In a still further aspect, the
method further comprises treatment with a vaccine. In a still further
aspect, the method further comprises treatment with an antibiotic. In a
still further aspect, the pathogen is a bacteria, and the method further
comprises the administration of an antibacterial agent. In a still
further aspect, the bacteria is selected from the group consisting of:
Mycobacterium spp., Salmonella spp., Listeria spp, Streptococcus spp.,
Haemophilus, spp., Neisseria spp., Klebsiella spp., Borrelia spp.,
Bacterioides fragillis, Treponema spp., and Helicobacter pylori. In a
still further aspect, the pathogen is a virus, and the method further
comprises the administration of an anti-viral agent. In a still further
aspect, the virus is selected from the group consisting of: hepatitis-B,
-C, herpes simplex virus-I, -II, human immunodeficiency virus-I, -II,
cytomegalovirus, Eppstein Barr virus, human papillomavirus, human T
lymphotrophic viruses, -I, -II, varicella zoster. In a still further
aspect, the pathogen is a fungus, and the method further comprises the
administration of an anti-fungal agent. In a still further aspect, the
disorder is selected from the group consisting of: aspergilosis,
blastomycosis, candidiasis albicans, coccidioiodmycosis immitis,
histoplasmosis, paracoccidioiomycosis, microsporidiosis. In a still
further aspect, the pathogen is a protozoan, and the method further
comprises the administration of an anti-protozoan agent. In a still
further aspect, the disorder is selected from the group consisting of:
leishmaniasis, plasmodiosis (i.e., malaria), cryptosporidiosis,
toxoplasmosis, trypanosomiasis, and helminth infections, including those
resulting from trematodes (e.g., schistosomiasis), cestodes (e.g.,
echinococcosis) and nemotodes (e.g., trchinosis, ascariasis, filariosis
and strongylodiosis).

[0038] In a still further aspect, the T-cell dysfunctional disorder is
tumor immunity. In a still further aspect, the PD-L1 antibody or
composition is combined with a treatment regimen further comprising a
traditional therapy selected from the group consisting of: radiation
therapy, chemotherapy, targeted therapy, immunotherapy, hormonal therapy,
angiogenesis inhibition and palliative care. In a still further specific
aspect, the chemotherapy treatment is selected from the group consisting
of: gemcitabine, cyclophosphamide, doxorubicin, paclitaxel, cisplatin. In
a still further specific aspect, the tumor immunity results from a cancer
selected from the group consisting of: breast, lung, colon, ovarian,
melanoma, bladder, kidney, liver, salivary, stomach, gliomas, thyroid,
thymic, epithelial, head and neck cancers, gastric, and pancreatic
cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] FIG. 1 is a graphical illustration depicting costimulation of
T-cells by the B7 family of cell surface molecules.

[0040]FIG. 2 is a schematic showing the experimental design of the
PMEL/B16 T-cell stimulation assay.

[0041]FIG. 3 is a bar graph showing the effect of anti-PD-L1 Ab on
antigen-specific T cell function through enhanced IFN-γ production
in PMEL CD8+ T cells in response to melanocyte peptide gp100. Both the
percentage of IFN-γ producing CD8+ T-cells and their levels of
IFN-γ production are increased during stimulation in the presence
of the anti-PD-L1 antibody.

[0042] FIG. 4 is a bar graph showing the effect of anti-PD-L1 Ab on
antigen-specific T cell function through enhancement in proliferation of
Ova-specific CD4+ T cells by the anti-PD-L1 Ab YW243.55.S1 in a secondary
stimulation with Ova-pulsed A20 B cells/mPD-L1 APCs.

[0043]FIG. 5 is a series of FACS plots showing the enhancement in
proliferation of human CD8 T cells by anti-PD-L1 antibody YW243.55S1 in a
Mixed Lymphocyte Reaction. The percent of proliferating cells as measured
by the dilution in intensity of CFSE is also reported.

[0044] FIG. 6 is a schematic of the experimental design of the treatment
of chronic LCMV with chimeric form of anti-PD-L1 Ab YW243.55S70. Arrows
designate the timing of the 6 doses of anti-PD-L1 begun 14 days post
infection with 2×106 pfu Clone 13 LCMV.

[0046] FIGS. 8A and 8B show the reduction in blood and tissue LCMV titers
in chronic LCMV infection following in vivo treatment with anti-PD-L1
antibody. In FIG. 8A, viral titers from the various indicated tissues are
analyzed at Days 21 and 28, one and two weeks after Ab treatment,
respectively. In FIG. 8B, serum viral titers are analyzed on Days 0, 7,
14, 21 and 28, with LCMV inoculation occurring on day 0 and treatment
commencing on day 14.

[0047]FIG. 9A shows a significant reduction in MC38.Ova colon carcinoma
tumor growth as a result of application of anti-PD-L1 antibody following
therapeutic treatment of established tumors (treatment begun at Day 14,
when tumor is 250 mm3) FIG. 9B is a histogram showing surface levels
of PD-L1 expression on MC38.Ova cells in tissue culture as measured by
flow cytometry. PD-L2 is not expressed by MC38.Ova cells.

[0048] FIG. 10 is a graph showing the effect of PD-L1 blockade treatment
alone and in combination with either anti-VEGF or Gemcitabine on the
growth of MC38.Ova tumors in C57BL/6 mice.

[0049] FIGS. 11A-1 to A-3 and FIGS. 11B-1 to B-3 are the heavy and light
chain variable region sequences, respectively, of 11 anti-PD-L1
antibodies identified by phage display. The shaded bars show CDRs with
various definitions, while the boxed areas show the extent of the HVRs.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0050] All references mentioned herein are specifically incorporated by
reference.

[0054] The two major types of lymphocytes in humans are T (thymus-derived)
and B (bone marrow derived. These cells are derived from hematopoietic
stem cells in the bone marrow and fetal liver that have committed to the
lymphoid development pathway. The progeny of these stem cells follow
divergent pathways to mature into either B or T lymphocytes. Human
B-lymphocyte development takes place entirely within the bone marrow. T
cells, on the other hand, develop from immature precursors that leave the
marrow and travel through the bloodstream to the thymus, where they
proliferate and differentiate into mature T lymphocytes.

[0055] Mature lymphocytes that emerge from the thymus or bone marrow are
in a quiescent, or "resting" state, i.e., they are mitotically inactive.
When dispersed into the bloodstream, these "naive" or "virgin"
lymphocytes, travel into various secondary or peripheral lymphoid organs,
such as the spleen, lymph nodes or tonsils. Most virgin lymphocytes have
an inherently short life span and die without a few days after leaving
the marrow or thymus. However, if such a cell receives signals that
indicate the presence of an antigen, they may activate and undergo
successive rounds of cell division. Some of the resulting progeny cells
then revert to the resting state to become memory lymphocytes--B and T
cells that are essentially primed for the next encounter with the
stimulating allergen. The other progeny of activated virgin lymphocytes
are effector cells, which survive for only a few days, but carry out
specific defensive activities.

[0056] Lymphocyte activation refers to an ordered series of events through
which a resting lymphocyte passes as it is stimulated to divide and
produce progeny, some of which become effector cells. A full response
includes both the induction of cell proliferation (mitogenesis) and the
expression of immunologic functions. Lymphocytes become activated when
specific ligands bind to receptors on their surfaces. The ligands are
different for T cells and B cells, but the resulting intracellular
physiological mechanisms are similar.

[0057] Some foreign antigens themselves can induce lymphocyte activation,
especially large polymeric antigens that cross-link surface
immunoglobulins on B-cells, or other glycoproteins on T-cells. However,
most antigens are not polymeric and even direct binding to B-cells in
large numbers fail to result in activation. These more common antigens
activate B cells when they are co-stimulated with nearby activated helper
T-lymphocytes. Such stimulation may occur from lymphokines secreted by
the T-cell, but is transmitted most efficiently by direct contact of the
B cell with T-cell surface proteins that interact with certain B-cell
surface receptors to generate a secondary signal.

[0058] B. T-Cells

[0059] T lymphocytes do not express immunoglobulins, but, instead detect
the presence of foreign substances by way of surface proteins called
T-cell receptors (TCR). These receptors recognize antigens by either
direct contact or through influencing the activity of other immune cells.
Together with macrophages, T cells are the primary cell type involved in
the cell-mediated immunity.

[0060] Unlike B-cells, T-cells can detect foreign substances only in
specific contexts. In particular, T-lymphocytes will recognize a foreign
protein only if it first cleaved into small peptides, which are then
displayed on the surface of a second host cell, called an
antigen-presenting cell (APC). Many types of host cells can present
antigens under some conditions but certain types are more specifically
adapted for this purpose and are particularly important in controlling
T-cell activity, including macrophages and other B-cells. Antigen
presentation depends in part on specific proteins, called major
histocompatibility complex (MHC) proteins, on the surface of the
presenting cells. Thus, to stimulate cell-mediated immunity, foreign
peptides must be presented to T-cells in combination with MHC peptides,
and this combination must be recognized by a T-cell receptor.

[0061] There are two significant T-cell subsets: cytotoxic T lymphocytes
(Tc cells or CTLs) and helper T cells (TH) cells, which can
roughly be identified on the basis of cell surface expression of the
marker CD8 and CD4. Tc cells are important in viral defense, and can
kill viruses directly by recognizing certain cell surface expressed viral
peptides. TH cells promote proliferation, maturation and immunologic
function of other cell types, e.g., lymphokine secretion to control
activities of B cells, macrophages and cytotoxic T cells. Both virgin and
memory T-lymphocytes ordinarily remain in the resting state, and in this
state they do not exhibit significant helper or cytotoxic activity. When
activated, these cells undergo several rounds of mitotic division to
produce daughter cells. Some of these daughter cells return to the
resting state as memory cells, but others become effector cells that
actively express helper or cytotoxic activity. These daughter cells
resemble their parents: CD4+ cells can only product CD4+ progeny, while
CD8+ cells yield only CD8+ progeny. Effector T-cells express cell surface
markers that are not expressed on resting T-cells, such as CD25, CD28,
CD29, CD40L, transferrin receptors and class II MHC proteins. When the
activating stimuli is withdrawn, cytotoxic or helper activity gradually
subsides over a period of several days as the effector cells either die
or revert to the resting state.

[0062] Similar to B-cell activation, T-lymphocyte responses to most
antigens also require two types of simultaneous stimuli. The first is the
antigen, which if appropriately displayed by MHC proteins on an
antigen-presenting cell, can be recognized and bound by T-cell receptors.
While this antigen-MHC complex does send a signal to the cell interior,
it is usually insufficient to result in T-cell activation. Full
activation, such as occurs with helper T-cells, requires costimulation
with other specific ligands called costimulators that are expressed on
the surface of the antigen-presenting cell. Activation of a cytotoxic T
cell, on the other hand, generally requires IL-2, a cytokine secreted by
activated helper T cells.

[0063] C. The Immune Response

[0064] The three primary functional properties of the mammalian immune
system distinguishing it from the other body's defenses include: (1)
specificity--the ability to recognize and respond or not to respond
individually among a vast number of target molecules, (2)
discrimination--the ability to determine self from non-self so as to
peacefully coexist with all the innumerable proteins and other organic
material, yet still respond vigorously against foreign material that is
introduced to the body, and (3) memory--the ability to be molded by
experience such that subsequent encounters with a particular foreign
pathogen will provoke a more rapid and vigorous response than what
occurred at the initial encounter. When one or more of these functions is
frustrated, a pathological condition results.

[0065] Virgin lymphocytes are continually released from the primary
lymphoid organs into the periphery, each carrying surface receptors that
enable antigen binding. Antigen binding in B cells is mediated through
surface-bound immunoglobulins, whereas in T-cells it is mediated by
T-cell receptors. When virgin lymphocytes are activated, they
proliferate, yielding daughter cells that may then undergo further cycles
of activation and proliferation. The speed and intensity of response to a
given antigen is determined largely by clonal selection: the larger the
population of daughter cells or clones specific to a particular antigen,
the greater the number of cells that can recognize and participate in the
immune response. Every immune response is complex and intricately
regulated sequence of events involving several cell types. It is
triggered when an immunogen enters the body and encounters a specialized
class of cells called antigen-presenting cells (APCs). These APCs capture
a minute amount of the immunogen and display it in a form that can be
recognized by antigen-specific helper T-lymphocytes. The helper T cells
then become activated and, in turn, promote activation of other classes
of lymphocytes, such as B cells or cytotoxic T cells. The activated
lymphocytes then proliferate and carry out their specific effector
functions. At each stage in this process, the lymphocytes and APCs
communicate with one another through direct contact or by secreting
regulatory cytokines.

[0066] Exogenous antigens that are captured by an APC undergo a series of
alterations called antigen processing. Such processing, especially of
proteinaceous immunogens involves denaturation and partial proteolytic
digestions, so that the immunogen is cleaved into short peptides. A
limited number of the resulting peptides then associated non-covalently
with class II MHC proteins and are transported to the APC surface, a
process known as antigen presentation. A CD4+ helper T lymphocyte that
comes into direct contact with an APC may become activated, but it will
do so only if it expressed a T-cell receptor protein that can recognize
and bind the particular peptide-MHC complex presented by the APC.

[0067] Helper T (TH) cells are the principal orchestrators of the
immune response because they are needed for activation of the two other
lymphatic effector cells: cytotoxic T (Tc) cells and antibody secreting
plasma cells. TH activation occurs early in an immune response and
requires at least two signals. One signal is provided by binding of the
T-cell antigen receptor to the antigenic peptide-MHC complex on the APC
surface that is transmitted through the CD3 protein complex, while the
second, costimulatory signal through the APC is thought to result from
binding of a separate signal-transmitting protein on the T-cell surface
with a specific ligand on the APC. One known such interaction is the
T-cell protein CD28 and the family of APC surface proteins known as B7.
Other surface proteins pairs may also mediate costimulation. The process
of co-stimulation is described in greater detail subsequently. The
anti-PD-L1 antibodies of the present invention are believed to enhance
co-stimulation through antagonisism of a negative costimulatory signal
provided by signaling through PD-L1.

[0068] Together, the two signals induce the helper T cell to begin
secreting the cytokine interleukin-2 (IL-2) and also to begin expressing
specific high affinity IL-2 receptors on its surface. IL-2 is a highly
potent mitogenic factor for T-lymphocytes and is essential for the
proliferative response of activated T-cells. The effect of IL-2 on the
cell from which it is secreted--a phenomenon known as an autocrine
effect. It has further been shown that even if a T-cell has received both
signals, it will not proliferate if its own surface IL-2 receptors are
blocked. IL-2 can also act on cells in the immediate vicinity, in a
so-called paracrine effect. This effect is especially important to
activate Tc cells, which generally do not produce enough IL-2 to
stimulate their own proliferation. In addition to IL-2, activated TH
cells secrete other cytokines and promote the growth, differentiation,
and functions of B-cells, macrophages and other cell types.

[0069] The contact between an APC and an antigen-specific TH cell
also has effect on the APC--one of the most important of which is the
release of IL-1. This cytokine is believed to act in an autocrine manner
to increase surface expression of class II MHC proteins and of various
adhesion molecules thereby strengthening the binding of the TH cell
and enhancing antigen presentation. At the same time, IL-1 functions in a
paracrine manner on the TH cell to promote IL-2 secretion and IL-2
receptor expression.

[0070] During activation of TH cells in the manner previously
described, some B-cells may also have been engaging the immunogen through
their antigen receptors, which are membrane-bound forms of the antibodies
that they will later secrete. Unlike T-cells, B-cells recognize an
immunogen in its free, unprocessed form. Specific antigen binding
provides one type of signal that can lead to B-cell activation. A second
type is provided by activated TH cells, which express proteins that
help activate the B cell by binding to non-immunoglobulin receptors on
its surface. These TH-derived signals, which act on any B cell
regardless of its antigen specificity, are known as helper factors. These
helper factors include IL-2, IL-4 and IL-6. However, help is more
efficiently achieved through cell-cell contact, which allows proteins on
the T-cell surface to directly contact those on the B cell. The most
effect form of contact-mediated help occurs when a protein called CD40
ligand (CD40L), which is expressed on TH cells only after they
become activated, binds to a protein called CD40 on B cells. In a process
known as by-stander activation, contact with an activated B cell can even
be sufficient to activate resting B cells even though its surface
immunoglobulins have not engaged in antigen.

[0071] Tc lymphocytes function to eradicate cells that express
foreign antigens on their surfaces, such as virus-infected host cells.
Most Tc cells express CD8 rather than CD4 and hence recognize
antigens in association with class I rather than class II MHC proteins.
When a somatic cell is infected by a virus, some immunogenic viral
proteins may undergo processing within the cell, and the resulting
peptides may then appear as surface complexes with class I MHC molecules.
These peptide-MHC complexes may then be recognized by the T-cell receptor
of an antigen-specific clone, providing one of two signals necessary for
Tc-cell activation. This first signal alone induces high-affinity
IL-2 receptors on the Tc cell. The second signal is furnished by
IL-2 secreted from a nearby activated TH lymphocyte. On receiving
both signals, the activated Tc cell acquires cytotoxic activity,
enabling it to kill the cell to which it is bound, as well as any other
cells bearing the same peptide-MHC class I complexes. In some cases,
killing occurs because the Tc releases specific toxins onto the
target cell; in others, the Tc induces the target cell to commit
suicide by apoptosis. The activated Tc cell also proliferates,
giving rise to additional Tc cells with the same antigen
specificity.

[0078] As a result, the antagonism of CTLA-4 (e.g., antagonist anti-CTLA
antibodies) and or agonizing B7.1/B7.2/CD28 may be useful to enhance
immune response in the treatment of infection (e.g., acute and chronic)
and tumor immunity.

[0079] 2. ICOS/ICOSL Signaling:

[0080] Another pathway of interaction between APC's and T-cells occurs
through ICOS (CD278) and ICOSL (B7-H2, CD275). ICOS/ICOSL signaling
promotes T-helper cell differentiation and effector function, and is
particularly important for interleukin-10 (IL-10) production, but plays a
more modest role in regulating T cell expansion and IL-2 production,
including regulatory T-cells, T cell tolerance and autoimmunity.

[0083] Another potential role for ICOS relates to sustaining TH1
responses. In an experimental model of autoimmune encephalomyelitis (EAE)
for multiple sclerosis, a TH1 disease mediated by myelin-specific
CD4.sup.+ T cells, shows that the outcome of ICOS blockade might be
distinct when costimulation is blocked during T-cell priming, then during
the effector phase of EAE. Dong et al., Nature 409: 97-101 (2001);
Rottman et al., Nature Immunol. 2: 605-611 (2001); Sporici et al., Clin.
Immunol. 100: 277-288 (2001). EAE induced by myelin oligodendrocyte
glycoprotein (MOG) is greatly exacerbated in ICOS.sup.-/- knock-out mice,
with increased production of IFN-γ compared to wild type.
Similarly, ICOS blockade during induction of EAE, exacerbated the disease
also resulting in increased IFN-γ production. Therefore, ICOS
blockade during priming leads to TH1 polarization of the response.
Interestingly, the priming of myelin-specific TCR transgenic T cells in
vitro in the presence of ICOS-Ig inhibited their ability to induce EAE,
in stark contrast to the results of ICOS-Ig blockade observed in vivo.
Sporici et al., supra. The difference for the opposing outcomes in vitro
and in vivo is not yet clear, but might reflect a role for ICOS on IL-10
producing regulatory T-cells, as well as effector T cells during ICOS
blockade in vivo. Co-stimulation through IL-10 is very effective at
enhancing IL-10 production and is more effective than co-stimulation
through CD28. Hutloff et al., supra. The IL-10, IL-12 regulatory loop is
critical in regulating EAE because IL-10-/-, but not IL4-/- mice develop
exacerbated EAE. Segal et al., J. Exp. Med. 187: 537-546 (1998).

[0089] 80: 3532-40 (2006). There are at least 4 variants of PD-1 that have
been cloned from activated human T cells, including transcripts lacking
(i) exon 2, (ii) exon 3, (iii) exons 2 and 3 or (iv) exons 2 through 4.
Nielsen et al., Cell. Immunol. 235: 109-16 (2005). With the exception of
PD-1Δex3, all variants are expressed at similar levels as full
length PD-1 in resting peripheral blood mononuclear cells (PBMCs).
Expression of all variants is significantly induced upon activation of
human T cells with anti-CD3 and anti-CD28. The PD-1Δex3 variants
lacks a transmembrane domain, and resembles soluble CTLA-4, which plays
an important role in autoimmunity Ueda et al., Nature 423: 506-11 (2003).
This variant is enriched in the synovial fluid and sera of patients with
rheumatoid arthritis. Wan et al., J. Immunol. 177: 8844-50 (2006).

[0095] Additionally, several studies show a receptor for PD-L1 or PD-L2
that is independent of PD-1. B7.1 has already been identified as a
binding partner for PD-L1. Butte et al., Immunity 27: 111-22 (2007).
Chemical crosslinking studies suggest that PD-L1 and B7.1 can interact
through their IgV-like domains. B7.1:PD-L1 interactions can induce an
inhibitory signal into T cells. Ligation of PD-L1 on CD4+ T cells by B7.1
or ligation of B7.1 on CD4+ T cells by PD-L1 delivers an inhibitory
signal. T cells lacking CD28 and CTLA-4 show decreased proliferation and
cytokine production when stimulated by anti-CD3 plus B7.1 coated beads.
In T cells lacking all the receptors for B7.1 (i.e., CD28, CTLA-4 and
PD-L1), T cell proliferation and cytokine production were no longer
inhibited by anti-CD3 plus B7.1 coated beads. This indicates that B7.1
acts specifically through PD-L1 on the T-cell in the absence of CD28 and
CTLA-4. Similarly, T cells lacking PD-1 showed decreased proliferation
and cytokine production when stimulated in the presence of anti-CD3 plus
PD-L1 coated beads, demonstrating the inhibitory effect of PD-L1 ligation
on B7.1 on T cells. When T cells lacking all known receptors for PD-L1
(i.e., no PD-1 and B7.1), T cell proliferation was no longer impaired by
anti-CD3 plus PD-L1 coated beads. Thus, PD-L1 can exert an inhibitory
effect on T cells either through B7.1 or PD-1.

[0096] The direct interaction between B7.1 and PD-L1 suggests that the
current understanding of costimulation is incomplete, and underscores the
significance to the expression of these molecules on T cells. Studies of
PD-L1.sup.-/- T cells indicate that PD-L1 on T cells can down-regulate T
cell cytokine production. Latchman et al., Proc. Natl. Acad. Sci. USA
101: 10691-96 (2004). Because both PD-L1 and B7.1 are expressed on T
cells, B cells, DCs and macrophages, there is the potential for
directional interactions between B7.1 and PD-L1 on these cells types.
Additionally, PD-L1 on non-hematopoietic cells may interact with B7.1 as
well as PD-1 on T cells, raising the question of whether PD-L1 is
involved in their regulation. One possible explanation for the inhibitory
effect of B7.1:PD-L1 interaction is that T cell PD-L1 may trap or
segregate away APC B7.1 from interaction with CD28.

[0097] As a result, the antagonism of signaling through PD-L1, including
blocking PD-L1 from interacting with either PD-1, B7.1 or both, thereby
preventing PD-L1 from sending a negative co-stimulatory signal to T-cells
and other antigen presenting cells is likely to enhance immunity in
response to infection (e.g., acute and chronic) and tumor immunity. In
addition, the anti-PD-L1 antibodies of the present invention, may be
combined with antagonists of other components of PD-1:PD-L1 signaling,
for example, antagonist anti-PD-1 and anti-PD-L2 antibodies.

[0100] Recent studies have shown that B7-H3 is both a stimulator and an
inhibitor of T cell responses. Evidence of stimulatory activation is
provided by the following: (1) In combination with anti-CD3, B7-H3/Ig
fusions costimulated CD4+ and CD8+ T cell proliferation, and stimulated
IFN-γ and CD8 lytic activity, Chapoval et al., Nat. Immunol. 2:
269-74 (2001); and (2) Injection of B7-H3 expression plasmid into tumors
of an EL-4 lymphoma model resulted in complete regression of 50% of
tumors, which was dependent upon CD8+ T cells and NK cells. However,
several recent studies have shown an inhibitory role for this molecule.
B7-H3.sup.-/- APC knockouts show a two-fold increase in alloreactive T
cell proliferation in an MLR response. Activation of CD4 T cells by
anti-CD3 and anti-CD28 was inhibited in HLA-DR2 transfected with either
form of B7-H3. Ling et al., Genomics 82: 365-77 (2003). The result was
reduced proliferation and production of IFN-γ, TNF-α, IL-10
and GM-CSF. The reconciliation of these studies could lie in the
existence of two receptors for B7-H3 with opposing functions, similar to
how CD28 and CTLA-4 regulate signaling via B7.1 and B7.2.

[0101] As a result, the blockade of B7-H3 signaling may contribute to
enhancing immune response to infection and tumor immunity when combined
with the anti-PD-L1 antibodies of the invention.

[0102] 5. B7-H4

[0103] The most recent addition to the B7 family is B7-H4 (B7x, B7-S1,
B7-H.5, VTCN1, PRO1291), which is a negative regulator of T cell
responses. Zang et al., Proc. Natl. Acad. Sci. U.S.A. 100 (18),
10388-10392 (2003); Watanabe et al., Nat. Immunol. 4 (7), 670-679 (2003);
Prasad, et al., Immunity 18(6), 863-873 (2003); Sica et al., Immunity 18
(6), 849-861 (2003). Both human and mouse B7-H4 are expressed broadly in
both lymphoid (spleen and thymus) and nonlymphoid organs (including lung,
liver, testis, ovary, placenta, skeletal muscle, pancreas and small
intestine). B7-H4 is not detected in normal human tissues by IHC or
regulation of B7-H4 at the translational level. IHC shows B7-H4 is highly
expressed in lung and ovarian tumors, and real-time polymerase chain
reaction (PCR) analysis indicate that mouse B7-H4 also is highly
expressed in prostate, lung and colon carcinoma cell lines. B7-H4 binds a
yet unknown receptor on activated, but not naive T cells that is distinct
from CTLA-4, ICOS, PD-1 and the receptor for B7-H3. Although BTLA was
initially reported to be the ligand for B7-H4, the reported binding of
B7-H4/Ig fusions to wild-type, but not BTLA.sup.-/- cells compels the
conclusion that HVEM, and not BTLA is the unique ligand for B7-H4. Sedy
et al., Nat. Immunol. 6: 90-98 (2004).

[0104] Studies with B7-H4 transfectants and immobilized B7-H4/Ig fusions
demonstrate that B7-H4 delivers a signal that inhibits TCR-mediated
CD4.sup.+ and CD8.sup.+ T cell proliferation, cell-cycle progression in
the G0/G1 phase, and IL-2 production. Sica et al., Immunity 18: 849-61
(2003); Zang et al., PNAS 100: 10388-92 (2003); Prasad et al., Immunity
18: 863-73 (2003). B7.1 costimulation cannot overcome B7-H4/Ig induced
inhibition. Blocking anti-B7-H4 antibody increased T cell proliferation
and IL-2 production in vitro. In vivo administration of anti-B7-H4
antibody commensurate with administration of kehole limpet hemacyanin
(KLH) in complete Freund's adjuvant (CFA) led to a modest increase in
anti-KLH antibody IgM production and a two- to three-fold increase in T
cell proliferation and IL-2 production upon in vitro restimulation with
KLH, suggesting greater T cell priming in vivo in the presence of
anti-B7-H4. Anti-B7-H4 blocking antibody markedly accelerated the onset
and severity of EAE in increased CD4.sup.+ and CD8.sup.+ T cells and
CD11b.sup.+ macrophages in the brain of anti-B7-H4 treated an autoimmune
mouse model. The combined experimental data available on B7-H4 suggest
that it may downregulate immune responses in peripheral tissues and play
a role in regulating T cell tolerance. The expression of B7-H4 may also
play a role in evasion of host immune responses in tumor immunity Choi et
al., J. Immunol. 171: 4650-54 (2003). As a result, the antagonism of
B7-H4 may be useful to enhance immune response to infection and tumor
immunity when combined with the anti-PD-L1 antibodies of the invention.

[0105] 6. BTLA:

[0106] The B7 family member BTLA (CD272, BTLA-1) is functionally similar
to PD-1 and CTLA. Initially identified as a selective marker for Th1
cells, BTLA is only expressed on lymphocytes. Similar to CTLA-4, ICOS and
PD-1, BTLA is induced on T cells during activation. However, in contrast
with ICOS, which remains elevated on Th2-cells, but is downregulated in
Th1 cells, BTLA remains expressed on Th1-cells, but not Th2-cells.
Similar to PD-1, BTLA is also expressed on B-cells. Gavrieli et al.,
Biochem. Biophys. Res. Commun. 312: 1236-43 (2003). However, BTLA is
expressed on both resting and activated B cells, whereas PD-1 is
upregulated on activated B cells. BTLA has two ITIM motifs.

[0107] BTLA exerts inhibitory effects on both B and T lymphocytes.
Watanabe et al., Nat. Immunol. 4: 670-79 (2003). BLTA.sup.-/- B cells
show modest response to anti-IgM, but an increased response to anti-CD3
in vitro. Polarized BTLA.sup.-/- Th1 cells show about a two-fold increase
in proliferation in response to antigen exposure, in vitro. In vivo,
BTLA.sup.-/- mice show a three-fold increase in hapten-specific antibody
responses and enhanced susceptibility to EAE. The phenotype of
BTLA.sup.-/- mice resembles the phenotype of PD-1.sup.-/- mice,
exhibiting increased susceptibility to autoimmunity, but more subtle
phenotypes than CTLA-4.sup.-/- mice. However, given its role as a
negative regulator, blockade of BTLA may prove useful for enhancing
immune response in infection and antitumor immunity when combined with
the anti-PD-L1 antibodies of the invention.

[0113] OX40 is believed to provide a late-acting signal that allow for the
survival of newly generated effector cells at the peak of the primary
immune response. There is also good evidence that OX40 functions
downstream from CD28--in addition to increased expression of OX40
mediated by CD28 signals, functional analysis of CD28 deficiency versus
OX40 deficiency have shown that early primary T-cell responses are
markedly impaired in the absence of CD28 signals, but only late responses
are impaired in the absence of OX40 signals. Rogers et al., Immunity 15:
445-455 (2001); Bertram et al., J. Immunol. 168: 3777-3785 (2002).

[0114] As a result, it is likely that activation of OX40/OX40L, such as
through the application of agonist antibodies may be useful when combined
with the anti-PD-L1 antibodies of the invention to treat T-cell
dysfunctional disorders.

[0117] Like OX40, 4-1BB is believed to provide a late-acting signal that
allow for the survival of newly generated effector cells at the peak of
the primary immune response. There is also good evidence that 4-1BB
functions later than CD28--in addition to increased expression of OX40
and 4-1BB mediated by CD28 signals, functional analysis of CD28
deficiency versus 4-1BB deficiency have shown that early primary T-cell
responses are markedly impaired in the absence of CD28 signals, but only
late responses are impaired in the absence of OX40 signals. Rogers et
al., Immunity 15: 445-455 (2001); Bertram et al., J. Immunol. 168:
3777-3785 (2002).

[0119] As a result, it is likely that activation of 4-1BB/4-BBL, such as
through the application of agonist antibodies, particularly in
combination with PD-L1 antagonists (e.g., anti-PD-L1 antibody) may be
useful to treat T-cell dysfunctional disorders.

[0120] 3. CD27/CD27L (CD70)

[0121] The importance of CD27 (TNFRSF7, 5152) and CD27L (CD70, TNFSF7)
signaling in the initial stages of a T-cell response has been
demonstrated in in vitro blocking studies, wherein CD27/CD70 interactions
were disrupted. Oshima et al., Int. Immunol. 10: 517-526 (1998); Agematsu
et al., J. Immunol. 153: 1421-1429 (1994); Hintzen et al., J. Immunol.
154: 2612-2623 (1995). T cells that lack CD27 initially divide normally,
but then proliferate poorly 3 or more days after activation. Hendriks et
al., Nature Immunol. 1: 433-440 (2000). This indicates that CD27
participates in promoting the initial expansion of the naive T-cell
population, by either early suppression of T-cell death or by acting on
the cell cycle to allow sustained division 2-3 days after activation.
This is reinforced by in vivo studies of CD27-deficient mice, in which
lower numbers of antigen-specific responses (days 4-8) and fewer memory T
cells develop over 3 or more weeks. Hendriks et al., supra. The
expression of CD27 is upregulated early after T-cell activation,
suggesting that it mainly delivers signals that maintain early
proliferation, before the peak of the effector response.

[0122] As a result, it is likely that activation of CD27/CD27L, including
through the application of agonist antibodies, particularly in
combination with the anti-PD-L1 antibodies described herein, may be
useful to treat T-cell dysfunctional disorders.

[0125] While the exact mechanisms of CD30/CD30L signaling is unclear, it
has been suggested that it might be similar to OX40 and 4-1BB. When
adoptively transferred antigen-specific CD8+ T cells are transferred into
CD30L-deficient mice, they do not accumulate in high numbers at the peak
of a primary response, and fewer memory T cells develop. As a result,
CD30 might also provide proliferation and/or survival signals to allow
the generation of high numbers of antigen-specific T cells at the peak of
primary responses.

[0126] As a result, it is likely that activation of CD27/CD27L, including
through the application of agonist antibodies, particularly in
combination with the anti-PD-L1 antibodies described herein, may be
useful to treat T-cell dysfunctional disorders.

[0129] As a result, the HVEM/LIGHT, such as through the application of
agonist antibodies, particularly in combination with the anti-PD-L1
antibodies described herein, may be useful to treat T-cell dysfunctional
disorders.

II. Definitions

[0130] An "allergen" or "immunogen` is any molecule that can trigger an
immune response. As used herein, the term covers either the antigenic
molecule itself, or its source, such as pollen grain, animal dander,
insect venom or food product. This is contrasted with the term antigen,
which refers to a molecule that can be specifically recognized by an
immunoglobulin or T-cell receptor.

[0131] Any foreign substance capable of inducing an immune response is a
potential allergen. Many different chemicals of both natural and
synthetic origin are known to be allergenic. Complex natural organic
chemicals, especially proteins, are likely to cause antibody-mediated
allergy, whereas simple organic compounds, inorganic chemicals, and
metals more preferentially cause T-cell mediated allergy. In some cases,
the same allergen may be responsible for more than one type of allergy.
Exposure to the allergen may be through inhalation, injection, injection,
or skin contact.

[0132] "Dysfunction" in the context of immune dysfunction, refers to a
state of immune reduced responsiveness to antigenic stimulation. The term
includes the common elements of both exhaustion and/or anergy in which
antigen recognition may occur, but the ensuing immune response is
ineffective to control infection or tumor growth.

[0133] "Tolerance" or "immunological tolerance" is the failure of the
immune system to mount a defensive immune response to a particular
antigen. Tolerance can be natural or self, wherein the body does not
attack its own proteins and antigens, or it can be induced, resulting
from the manipulation of the immune system. Central tolerance occurs
during lymphocyte development and operates in the thymus and bone marrow.
During this process, T and B lymphocytes that recognize self antigens are
deleted before they develop into fully immunocompetent cells. This
process is most active during fetal development, but continues throughout
life as immature lymphocytes are generated. Peripheral T-cell tolerance
refers to a functional unresponsiveness to self-antigens that are present
in peripheral tissues, and occurs after T and B cells mature and enter
the periphery. These processes include the suppression of autoreactive
cells by "regulatory" T cells and the generation of hyporesponsiveness
(anergy) in lymphocytes which encounter antigen in the absence of the
co-stimulatory signals that accompany inflammation. "Acquired" or
"induced tolerance" refers to the immune system's adaptation to external
antigens characterized by a specific non-reactivity of the lymphoid
tissues to a given antigen that in other circumstances would likely
induce cell-mediated or humoral immunity. In adults, tolerance may be
clinically induced by repeated administration of very large doses of
antigen, or of small doses that are below the threshold required for
stimulation of an immune response, such as via intravenous or sublingual
administration of soluble antigens. Immunosuppression also facilitates
the induction of tolerance. The breakdown of self tolerance can lead to
autoimmunity.

[0134] "Enhancing T-cell function" means to induce, cause or stimulate a
T-cell to have a sustained or amplified biological function, or renew or
reactivate exhausted or inactive T-cells. Examples of enhancing T-cell
function include: increased secretion of γ-interferon from
CD8.sup.+ T-cells, increased proliferation, increased antigen
responsiveness (e.g., viral or pathogen clearance) relative to such
levels before the intervention. In one embodiment, the level of
enhancement is as least 50%, alternatively 60%, 70%, 80%, 90%, 100%,
120%, 150%, 200%. The manner of measuring this enhancement is known to
one of ordinary skill in the art.

[0135] A "T cell dysfunctional disorder" is a disorder or condition of
T-cells characterized by decreased responsiveness to antigenic
stimulation. In a particular embodiment, a T-cell dysfunctional disorder
is a disorder that is specifically associated with inappropriate
increased signaling through PD-1. In another embodiment, T-cell
dysfunctional disorder is one in which T-cells are anergic or have
decreased ability to secrete cytokines, proliferate, or execute cytolytic
activity. In a specific aspect, the decreased responsiveness results in
ineffective control of a pathogen or tumor expressing an immunogen.
Examples of T cell dysfunctional disorders characterized by T-cell
dysfunction include unresolved acute infection, chronic infection and
tumor immunity.

[0136] "Chronic infection" refers to an infection in which an infectious
agent (e.g., pathogens such as viruses, bacteria, protozoan parasites,
fungi, or the like) has induced an immune response in the infected host,
but has not been cleared or eliminated from that host as during an acute
infection. Chronic infections may be persistent, latent, or slow. While
acute infections are typically resolved by the immune system within a few
days or weeks (e.g., influenza), persistent infections can persist at a
relatively low level for months, years, decades, or a lifetime (e.g.,
Hepatitis B). In contrast, a latent infection is characterized by a long
period of asymptomatic activity punctuated by a period of rapidly
increasing high grade infection and elevated pathogen levels (e.g.,
herpes simplex). Finally, a slow infection is one characterized by a
gradual and continuous increase in disease symptoms, such as a long
period of incubation followed by a protracted and progressive clinical
course beginning after the onset of clinical symptoms. Unlike latent and
persistent infections, slow infection may not begin with an acute period
of viral multiplication (e.g., picornaviruses infection, visnavirus,
scrapie, Creutzfeldt-Jakob disease). Exemplary infectious agents capable
of inducing a chronic infection include viruses (e.g., cytomegalovirus,
Epstein Barr virus, hepatitis B virus, hepatitis C virus, herpes simplex
virus, types I and II, human immunodeficiency virus, types 1 and 2, human
papillomavirus, human T lymphotrophic viruses, types 1 and 2, varicella
zoster virus and the like), bacteria (e.g., Mycobacterium tuberculosis,
Listeria spp., Klebsiella pneumoniae, Streptococcus pneumoniae,
Staphylococcus aureus, Borrelia spp., Helicobacter pylori, and the like),
protozoan parasites (e.g., Leishmania spp., Plasmodium falciparum,
Schistosoma spp., Toxoplasma spp., Trypanosoma spp., Taenia carssiceps
and the like), and fungi (e.g., Aspergillus spp., Candida albicans,
Coccidioides immitis, Histoplasma capsulatum, Pneumocystis carinii and
the like). Additional infectious agents include prions or misfolded
proteins that affect the brain or neuron structure by further propagating
protein misfolding in these tissues, resulting in the formation of
amyloid plaques which cause cell death, tissue damage and eventual death.
Example of disease resulting from prion infection include:
Creutzfeldt-Jakob disease and its varieties,
Gerstmann-Straussler-Scheinker syndrome (GSS), fatal familial insomnia
(sFI), kuru, scrapie, Bovine spongiform encephalopathy (BSE) in cattle
(aka "mad cow" disease), and various other animal forms of encephalopathy
[e.g., transmissible mink encephalopathy (TME), chronic wasting disease
(CWD) in white-tailed deer, elk and mule deer, feline spongiform
encephalopathy, exotic ungulate encephalopathy (EUE) in nyala, oryx and
greater kudu, spongiform encephalopathy of the ostrich].

[0137] "Tumor immunity" refers to the process in which tumors evade immune
recognition and clearance. Thus, as a therapeutic concept, tumor immunity
is "treated" when such evasion is attenuated, and the tumors are
recognized and attacked by the immune system. Examples of tumor
recognition include tumor binding, tumor shrinkage and tumor clearance.

[0138] A "B 7-negative costimulatory antagonist" ("BNCA") is an agent that
decreases, blocks, inhibits, abrogates or interferes with the negative
co-stimulatory signal mediated by or through cell surface proteins
expressed on T lymphocytes mediated by a member of the B7 family. In one
aspect, a BNCA may either alone, or in combination with the anti-PD-1
antibodies of the invention render a dysfunctional T-cell
non-dysfunctional. In another aspect, a BNCA may be an agent that
inhibits nucleic acid or protein synthesis, expression, signaling, and/or
post-expression processing of a B7-negative costimulatory molecule. In
yet another aspect, a BNCA is an antibody, antigen binding antibody
fragment, BNCA oligopeptide, BNCA RNAi or BNCA small molecule that
decreases, blocks, inhibits, abrogates or interferes with signal
transduction by a B7-negative costimulatory molecule. Example B7 negative
costimulatory molecules includes: CTLA-4, PD-L1, PD-1, B7.1 (expressed on
T-cells), PD-L2, B7-H3 and B7-H4.

[0139] A positive costimulatory agonist is a molecule that increases,
enhances, augments or facilitates a co-stimulatory signal mediated by or
through cell surface proteins expressed on T lymphocytes. In one aspect,
a positive costimulatory molecule can be an extracellular domain, soluble
construct or agonist antibody which activates a positive costimulatory
pathway. Example positive costimulatory molecules include the B7
superfamily molecules, e.g., B7.1, B7.2, CD28 and ICOS/ICOSL. Additional
examples include the TNFR family costimulatory molecules, e.g.,
OX40/OX40L, 41-BB/41-BBL, CD27/CD27L, CD30/CD30L and HVEM/LIGHT.

[0140] A "small molecule" or "small organic molecule" is one that has a
molecular weight below about 500 Daltons.

[0141] An "interfering RNA" "RNAi" is RNA of 10 to 50 nucleotides in
length which reduces expression of a target gene, wherein portions of the
strand are sufficiently complementary (e.g., having at least 80% identity
to the target gene). The method of RNA interference refers to the
target-specific suppression of gene expression (i.e., "gene silencing"),
occurring at a post-transcriptional level (e.g., translation), and
includes all posttranscriptional and transcriptional mechanisms of RNA
mediated inhibition of gene expression, such as those described in P. D.
Zamore, Science 296: 1265 (2002) and Hannan and Rossi, Nature 431:
371-378 (2004). As used herein, RNAi can be in the form of small
interfering RNA (siRNA), short hairpin RNA (shRNA), and/or micro RNA
(miRNA). Such RNAi molecules are often a double stranded RNA complexes
that may be expressed in the form of separate complementary or partially
complementary RNA strands. Methods are well known in the art for
designing double-stranded RNA complexes. For example, the design and
synthesis of suitable shRNA and siRNA may be found in Sandy et al.,
BioTechniques 39: 215-224 (2005).

[0142] A "small interfering RNA" or siRNA is a double stranded RNA (dsRNA)
duplex of 10 to 50 nucleotides in length which reduces expression of a
target gene, wherein portions of the first strand is sufficiently
complementary (e.g., having at least 80% identity to the target gene).
siRNAs are designed specifically to avoid the anti-viral response
characterized by elevated interferon synthesis, nonspecific protein
synthesis inhibition and RNA degradation that often results in suicide or
death of the cell associated with the use of RNAi in mammalian cells.
Paddison et al., Proc Natl Acad Sci USA 99(3): 1443-8. (2002).

[0143] The term "hairpin" refers to a looping RNA structure of 7-20
nucleotides. A "short hairpin RNA" or shRNA is a single stranded RNA 10
to 50 nucleotides in length characterized by a hairpin turn which reduces
expression of a target gene, wherein portions of the RNA strand are
sufficiently complementary (e.g., having at least 80% identity to the
target gene). The term "stem-loop" refers to a pairing between two
regions of the same molecule base-pair to form a double helix that ends
in a short unpaired loop, giving a lollipop-shaped structure.

[0144] A "micro RNA" or "miRNA" (previously known as stRNA) is a single
stranded RNA of about 10 to 70 nucleotides in length that are initially
transcribed as pre-miRNA characterized by a "stem-loop" structure, which
are subsequently processed into mature miRNA after further processing
through the RNA-induced silencing complex (RISC).

[0145] A "BNCA interfering RNA" or "BNCA RNAi" binds, preferably
specifically, to a BNCA nucleic acid and reduces its expression. This
means the expression of the B7 negative costimulatory molecule is lower
with the BNCA RNAi present as compared to expression of the B7 negative
costimulatory molecule in a control where the BNCA RNAi is not present.
BNCA RNAi may be identified and synthesized using known methods (Shi Y.,
Trends in Genetics 19(1): 9-12 (2003), WO2003056012, WO2003064621,
WO2001/075164, WO2002/044321.

[0147] A "BNCA small molecule antagonist" or "BNCA small molecule" is an
organic molecule other than an oligopeptide or antibody as defined herein
that inhibits, preferably specifically, a B7 negative costimulatory
polypeptide. Such B7 negative co-stimulatory signaling inhibition
preferably renders a dysfunctional T-cell responsive to antigen
stimulation. Example BNCA small molecules may be identified and
chemically synthesized using known methodology (see, e.g., PCT
Publication Nos. WO2000/00823 and WO2000/39585). Such BNCA small
molecules are usually less than about 2000 daltons in size, alternatively
less than about 1500, 750, 500, 250 or 200 daltons in size, are capable
of binding, preferably specifically, to a B7 negative stimulatory
polypeptide as described herein, and may be identified without undue
experimentation using well known techniques. In this regard, it is noted
that techniques for screening organic molecule libraries for molecules
that are capable of binding to a polypeptide target are well known in the
art (see, e.g., PCT Publication Nos. WO00/00823 and WO00/39585).

[0152] The term "vaccine" as used herein includes any nonpathogenic
immunogen that, when inoculated into a host, induces protective immunity
against a specific pathogen. Vaccines can take many forms. Vaccines can
be whole organisms that share important antigens with the pathogem, but
are not pathogenic themselves (e.g., cowpox). Vaccines can also be
prepared from killed (e.g., Salk polio vaccine) or attenuated (lost
ability to produce disease--e.g., Sabin polio vaccine). Vaccines can also
be prepared from purified macromolecules isolated from the pathogenic
organism. For example, toxoid vaccines (e.g., tetanus and diphtheria)
containing the inactive form of soluble bacterial toxin--resulting in the
production of anti-toxin antibodies, but not immunity to the intact
bacteria. Subunit vaccines (e.g., Hepatitis B) contain only a single
immunogenic protein isolated from the pathogen of interest. Hapten
conjugate vaccines attaches certain carbohydrate or polypeptide epitopes
isolated from the pathogen of interest to immunogenic carriers, such as
tetanus toxoid. These strategies essentially use the epitopes as haptens
to induce antibody production, which then recognize the same epitope in
the native pathogen. However, to be maximally effective, such vaccines
must incorporate both B- and T-cell epitopes, and the T-cell epitopes
must be chosen to ensure that they can be recognized, presented and
responded to by the immune systems of the host individuals.

[0153] DNA vaccines exploit the ability of host cells to take up and
express DNA encoding pathogenic proteins that is injected
intramuscularly.

[0154] Examples of anti-viral vaccines that can be used in combination
with the anti-PD-L1 antibodies for the methods described herein include:
HCV vaccine (virasome) by Pevion Biotech., TG4040 (MVA-HCV by Transgene
viron designed to enhance cellular (Cytotoxic T lymphocytes CD4+and CD8+)
immune response against NS3, NS4 and NS5B, CHRONVAC®--a
codon-optimized NS3/4a DNA vaccine by Inovio Biomedical, HCV/CpG vaccines
by Novartis, GI-5005--an HCV vaccine by Globeimmune, IC41 a mixture of
synthetic peptides having HCV CD4 and CD8 T epitopes in combination with
poly-L-arginine by Intercell.

[0155] Host responses to immunogens can be enhanced if administered as a
mixture with adjuvants. Immune adjuvants function in one or more of the
following ways: (1) prolonging retention of the immunogen, (2) increased
effective size of the immunogen (and hence promoting phagocytosis and
presentation to macrophages), (3) stimulating the influx of macrophage or
other immune cells to the injection site, or (4) promoting local cytokine
production and other immunologic activities. Example adjuvants include:
complete Freund's adjuvant (CFA), aluminum salts, and mycobacterial
derived proteins such as muramyl di- or tri-peptides.

[0156] The term "antibody" includes monoclonal antibodies (including full
length antibodies which have an immunoglobulin Fc region), antibody
compositions with polyepitopic specificity, multispecific antibodies
(e.g., bispecific antibodies, diabodies, and single-chain molecules, as
well as antibody fragments (e.g., Fab, F(ab')2, and Fv). The term
"immunoglobulin" (Ig) is used interchangeably with "antibody" herein.

[0157] The basic 4-chain antibody unit is a heterotetrameric glycoprotein
composed of two identical light (L) chains and two identical heavy (H)
chains. An IgM antibody consists of 5 of the basic heterotetramer units
along with an additional polypeptide called a J chain, and contains 10
antigen binding sites, while IgA antibodies comprise from 2-5 of the
basic 4-chain units which can polymerize to form polyvalent assemblages
in combination with the J chain. In the case of IgGs, the 4-chain unit is
generally about 150,000 daltons. Each L chain is linked to an H chain by
one covalent disulfide bond, while the two H chains are linked to each
other by one or more disulfide bonds depending on the H chain isotype.
Each H and L chain also has regularly spaced intrachain disulfide
bridges. Each H chain has at the N-terminus, a variable domain (VH)
followed by three constant domains (CH) for each of the α and
γ chains and four CH domains for μ and ε isotypes.
Each L chain has at the N-terminus, a variable domain (VL) followed
by a constant domain at its other end. The VL is aligned with the
VH and the CL is aligned with the first constant domain of the
heavy chain (CH1). Particular amino acid residues are believed to
form an interface between the light chain and heavy chain variable
domains. The pairing of a VH and VL together forms a single
antigen-binding site. For the structure and properties of the different
classes of antibodies, see e.g., Basic and Clinical Immunology, 8th
Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds),
Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6. The L
chain from any vertebrate species can be assigned to one of two clearly
distinct types, called kappa and lambda, based on the amino acid
sequences of their constant domains. Depending on the amino acid sequence
of the constant domain of their heavy chains (CH), immunoglobulins can be
assigned to different classes or isotypes. There are five classes of
immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains
designated α, δ, ε, γ and μ, respectively.
The γ and α classes are further divided into subclasses on
the basis of relatively minor differences in the CH sequence and
function, e.g., humans express the following subclasses: IgG1, IgG2A,
IgG2B, IgG3, IgG4, IgA1 and IgA2.

[0158] An "isolated" antibody is one that has been identified, separated
and/or recovered from a component of its production environment (E.g.,
natural or recombinant). Preferably, the isolated polypeptide is free of
association with all other components from its production environment.
Contaminant components of its production environment, such as that
resulting from recombinant transfected cells, are materials that would
typically interfere with research, diagnostic or therapeutic uses for the
antibody, and may include enzymes, hormones, and other proteinaceous or
non-proteinaceous solutes. In preferred embodiments, the polypeptide will
be purified: (1) to greater than 95% by weight of antibody as determined
by, for example, the Lowry method, and in some embodiments, to greater
than 99% by weight; (1) to a degree sufficient to obtain at least 15
residues of N-terminal or internal amino acid sequence by use of a
spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under
non-reducing or reducing conditions using Coomassie blue or, preferably,
silver stain. Isolated antibody includes the antibody in situ within
recombinant cells since at least one component of the antibody's natural
environment will not be present. Ordinarily, however, an isolated
polypeptide or antibody will be prepared by at least one purification
step.

[0159] The "variable region" or "variable domain" of an antibody refers to
the amino-terminal domains of the heavy or light chain of the antibody.
The variable domains of the heavy chain and light chain may be referred
to as "VH" and "VL", respectively. These domains are generally the most
variable parts of the antibody (relative to other antibodies of the same
class) and contain the antigen binding sites.

[0160] The term "variable" refers to the fact that certain segments of the
variable domains differ extensively in sequence among antibodies. The V
domain mediates antigen binding and defines the specificity of a
particular antibody for its particular antigen. However, the variability
is not evenly distributed across the entire span of the variable domains.
Instead, it is concentrated in three segments called hypervariable
regions (HVRs) both in the light-chain and the heavy chain variable
domains. The more highly conserved portions of variable domains are
called the framework regions (FR). The variable domains of native heavy
and light chains each comprise four FR regions, largely adopting a
beta-sheet configuration, connected by three HVRs, which form loops
connecting, and in some cases forming part of, the beta-sheet structure.
The HVRs in each chain are held together in close proximity by the FR
regions and, with the HVRs from the other chain, contribute to the
formation of the antigen binding site of antibodies (see Kabat et al.,
Sequences of Immunological Interest, Fifth Edition, National Institute of
Health, Bethesda, Md. (1991)). The constant domains are not involved
directly in the binding of antibody to an antigen, but exhibit various
effector functions, such as participation of the antibody in
antibody-dependent cellular toxicity.

[0162] The term "naked antibody" refers to an antibody that is not
conjugated to a cytotoxic moiety or radiolabel.

[0163] The terms "full-length antibody," "intact antibody" or "whole
antibody" are used interchangeably to refer to an antibody in its
substantially intact form, as opposed to an antibody fragment.
Specifically whole antibodies include those with heavy and light chains
including an Fc region. The constant domains may be native sequence
constant domains (e.g., human native sequence constant domains) or amino
acid sequence variants thereof. In some cases, the intact antibody may
have one or more effector functions.

[1995]); single-chain antibody molecules and multispecific antibodies
formed from antibody fragments. Papain digestion of antibodies produced
two identical antigen-binding fragments, called "Fab" fragments, and a
residual "Fc" fragment, a designation reflecting the ability to
crystallize readily. The Fab fragment consists of an entire L chain along
with the variable region domain of the H chain (VH), and the first
constant domain of one heavy chain (CH1). Each Fab fragment is
monovalent with respect to antigen binding, i.e., it has a single
antigen-binding site. Pepsin treatment of an antibody yields a single
large F(ab')2 fragment which roughly corresponds to two disulfide
linked Fab fragments having different antigen-binding activity and is
still capable of cross-linking antigen. Fab' fragments differ from Fab
fragments by having a few additional residues at the carboxy terminus of
the CH1 domain including one or more cysteines from the antibody
hinge region. Fab'-SH is the designation herein for Fab' in which the
cysteine residue(s) of the constant domains bear a free thiol group.
F(ab')2 antibody fragments originally were produced as pairs of Fab'
fragments which have hinge cysteines between them. Other chemical
couplings of antibody fragments are also known.

[0165] The Fc fragment comprises the carboxy-terminal portions of both H
chains held together by disulfides. The effector functions of antibodies
are determined by sequences in the Fc region, the region which is also
recognized by Fc receptors (FcR) found on certain types of cells.

[0166] "Fv" is the minimum antibody fragment which contains a complete
antigen-recognition and -binding site. This fragment consists of a dimer
of one heavy- and one light-chain variable region domain in tight,
non-covalent association. From the folding of these two domains emanate
six hypervariable loops (3 loops each from the H and L chain) that
contribute the amino acid residues for antigen binding and confer antigen
binding specificity to the antibody. However, even a single variable
domain (or half of an Fv comprising only HVRs specific for an antigen)
has the ability to recognize and bind antigen, although at a lower
affinity than the entire binding site.

[0167] "Single-chain Fv" also abbreviated as "sFv" or "scFv" are antibody
fragments that comprise the VH and VL antibody domains
connected into a single polypeptide chain. Preferably, the sFv
polypeptide further comprises a polypeptide linker between the VH
and VL domains which enables the sFv to form the desired structure
for antigen binding. For a review of the sFv, see Pluckthun in The
Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore
eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0168] "Functional fragments" of the antibodies of the invention comprise
a portion of an intact antibody, generally including the antigen binding
or variable region of the intact antibody or the Fc region of an antibody
which retains or has modified FcR binding capability. Examples of
antibody fragments include linear antibody, single-chain antibody
molecules and multispecific antibodies formed from antibody fragments.

[0169] The term "diabodies" refers to small antibody fragments prepared by
constructing sFv fragments (see preceding paragraph) with short linkers
(about 5-10) residues) between the VH and VL domains such that
inter-chain but not intra-chain pairing of the V domains is achieved,
thereby resulting in a bivalent fragment, i.e., a fragment having two
antigen-binding sites. Bispecific diabodies are heterodimers of two
"crossover" sFv fragments in which the VH and VL domains of the
two antibodies are present on different polypeptide chains. Diabodies are
described in greater detail in, for example, EP 404,097; WO 93/11161;
Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).

[0170] The monoclonal antibodies herein specifically include "chimeric"
antibodies (immunoglobulins) in which a portion of the heavy and/or light
chain is identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a particular
antibody class or subclass, while the remainder of the chain(s) is(are)
identical with or homologous to corresponding sequences in antibodies
derived from another species or belonging to another antibody class or
subclass, as well as fragments of such antibodies, so long as they
exhibit the desired biological activity (U.S. Pat. No. 4,816,567;
Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
Chimeric antibodies of interest herein include PRIMATIZED® antibodies
wherein the antigen-binding region of the antibody is derived from an
antibody produced by, e.g., immunizing macaque monkeys with an antigen of
interest. As used herein, "humanized antibody" is used a subset of
"chimeric antibodies."

[0171] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies that contain minimal sequence derived from non-human
immunoglobulin. In one embodiment, a humanized antibody is a human
immunoglobulin (recipient antibody) in which residues from an HVR
(hereinafter defined) of the recipient are replaced by residues from an
HVR of a non-human species (donor antibody) such as mouse, rat, rabbit or
non-human primate having the desired specificity, affinity, and/or
capacity. In some instances, framework ("FR") residues of the human
immunoglobulin are replaced by corresponding non-human residues.
Furthermore, humanized antibodies may comprise residues that are not
found in the recipient antibody or in the donor antibody. These
modifications may be made to further refine antibody performance, such as
binding affinity. In general, a humanized antibody will comprise
substantially all of at least one, and typically two, variable domains,
in which all or substantially all of the hypervariable loops correspond
to those of a non-human immunoglobulin sequence, and all or substantially
all of the FR regions are those of a human immunoglobulin sequence,
although the FR regions may include one or more individual FR residue
substitutions that improve antibody performance, such as binding
affinity, isomerization, immunogenicity, etc. The number of these amino
acid substitutions in the FR are typically no more than 6 in the H chain,
and in the L chain, no more than 3. The humanized antibody optionally
will also comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin. For further
details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et
al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.
2:593-596 (1992). See also, for example, Vaswani and Hamilton, Ann.
Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc.
Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech.
5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409.

[0172] A "human antibody" is an antibody that possesses an amino-acid
sequence corresponding to that of an antibody produced by a human and/or
has been made using any of the techniques for making human antibodies as
disclosed herein. This definition of a human antibody specifically
excludes a humanized antibody comprising non-human antigen-binding
residues. Human antibodies can be produced using various techniques known
in the art, including phage-display libraries. Hoogenboom and Winter, J.
Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991).
Also available for the preparation of human monoclonal antibodies are
methods described in Cole et al., Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol.,
147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin.
Pharmacol., 5: 368-74 (2001). Human antibodies can be prepared by
administering the antigen to a transgenic animal that has been modified
to produce such antibodies in response to antigenic challenge, but whose
endogenous loci have been disabled, e.g., immunized xenomice (see, e.g.,
U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE®
technology). See also, for example, Li et al., Proc. Natl. Acad. Sci.
USA, 103:3557-3562 (2006) regarding human antibodies generated via a
human B-cell hybridoma technology.

[0173] The term "hypervariable region," "HVR," or "HV," when used herein
refers to the regions of an antibody variable domain which are
hypervariable in sequence and/or form structurally defined loops.
Generally, antibodies comprise six HVRs; three in the VH (H1, H2, H3),
and three in the VL (L1, L2, L3). In native antibodies, H3 and L3 display
the most diversity of the six HVRs, and H3 in particular is believed to
play a unique role in conferring fine specificity to antibodies. See,
e.g., Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, in Methods in
Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003).
Indeed, naturally occurring camelid antibodies consisting of a heavy
chain only are functional and stable in the absence of light chain. See,
e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al.,
Nature Struct. Biol. 3:733-736 (1996).

[0174] A number of HVR delineations are in use and are encompassed herein.
The Kabat Complementarity Determining Regions (CDRs) are based on
sequence variability and are the most commonly used (Kabat et al.,
Sequences of Proteins of Immunological Interest, 5th Ed. Public Health
Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia
refers instead to the location of the structural loops (Chothia and Lesk,
J. Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromise
between the Kabat HVRs and Chothia structural loops, and are used by
Oxford Molecular's AbM antibody modeling software. The "contact" HVRs are
based on an analysis of the available complex crystal structures. The
residues from each of these HVRs are noted below.

[0175] HVRs may comprise "extended HVRs" as follows: 24-36 or 24-34 (L1),
46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (H1),
50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. The
variable domain residues are numbered according to Kabat et al., supra,
for each of these definitions.

[0176] The expression "variable-domain residue-numbering as in Kabat" or
"amino-acid-position numbering as in Kabat," and variations thereof,
refers to the numbering system used for heavy-chain variable domains or
light-chain variable domains of the compilation of antibodies in Kabat et
al., supra. Using this numbering system, the actual linear amino acid
sequence may contain fewer or additional amino acids corresponding to a
shortening of, or insertion into, a FR or HVR of the variable domain. For
example, a heavy-chain variable domain may include a single amino acid
insert (residue 52a according to Kabat) after residue 52 of H2 and
inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to
Kabat) after heavy-chain FR residue 82. The Kabat numbering of residues
may be determined for a given antibody by alignment at regions of
homology of the sequence of the antibody with a "standard" Kabat numbered
sequence.

[0177] "Framework" or "FR" residues are those variable-domain residues
other than the HVR residues as herein defined.

[0178] A "human consensus framework" or "acceptor human framework" is a
framework that represents the most commonly occurring amino acid residues
in a selection of human immunoglobulin VL or VH framework sequences.
Generally, the selection of human immunoglobulin VL or VH sequences is
from a subgroup of variable domain sequences. Generally, the subgroup of
sequences is a subgroup as in Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991). Examples include for the VL,
the subgroup may be subgroup kappa I, kappa II, kappa III or kappa IV as
in Kabat et al., supra. Additionally, for the VH, the subgroup may be
subgroup I, subgroup II, or subgroup III as in Kabat et al., supra.
Alternatively, a human consensus framework can be derived from the above
in which particular residues, such as when a human framework residue is
selected based on its homology to the donor framework by aligning the
donor framework sequence with a collection of various human framework
sequences. An acceptor human framework "derived from" a human
immunoglobulin framework or a human consensus framework may comprise the
same amino acid sequence thereof, or it may contain pre-existing amino
acid sequence changes. In some embodiments, the number of pre-existing
amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or
less, 5 or less, 4 or less, 3 or less, or 2 or less.

[0181] An "amino-acid modification" at a specified position, e.g. of the
Fc region, refers to the substitution or deletion of the specified
residue, or the insertion of at least one amino acid residue adjacent the
specified residue. Insertion "adjacent" to a specified residue means
insertion within one to two residues thereof. The insertion may be
N-terminal or C-terminal to the specified residue. The preferred amino
acid modification herein is a substitution.

[0182] An "affinity-matured" antibody is one with one or more alterations
in one or more HVRs thereof that result in an improvement in the affinity
of the antibody for antigen, compared to a parent antibody that does not
possess those alteration(s). In one embodiment, an affinity-matured
antibody has nanomolar or even picomolar affinities for the target
antigen. Affinity-matured antibodies are produced by procedures known in
the art. For example, Marks et al., Bio/Technology 10:779-783 (1992)
describes affinity maturation by VH- and VL-domain shuffling. Random
mutagenesis of HVR and/or framework residues is described by, for
example: Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994);
Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol.
155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995);
and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).

[0183] As use herein, the term "specifically binds to" or is "specific
for" refers to measurable and reproducible interactions such as binding
between a target and an antibody, which is determinative of the presence
of the target in the presence of a heterogeneous population of molecules
including biological molecules. For example, an antibody that
specifically binds to a target (which can be an epitope) is an antibody
that binds this target with greater affinity, avidity, more readily,
and/or with greater duration than it binds to other targets. In one
embodiment, the extent of binding of an antibody to an unrelated target
is less than about 10% of the binding of the antibody to the target as
measured, e.g., by a radioimmunoassay (RIA). In certain embodiments, an
antibody that specifically binds to a target has a dissociation constant
(Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, or
≦0.1 nM. In certain embodiments, an antibody specifically binds to
an epitope on a protein that is conserved among the protein from
different species. In another embodiment, specific binding can include,
but does not require exclusive binding.

[0184] A "blocking" antibody or an "antagonist" antibody is one that
inhibits or reduces a biological activity of the antigen it binds. In
some embodiments, blocking antibodies or antagonist antibodies
substantially or completely inhibit the biological activity of the
antigen. The anti-PD-L1 antibodies of the invention block the signaling
through PD-1 so as to restore a functional response by T-cells from a
dysfunctional state to antigen stimulation.

[0185] An "agonist" or activating antibody is one that enhances or
initiates signaling by the antigen to which it binds. In some
embodiments, agonist antibodies cause or activate signaling without the
presence of the natural ligand.

[0186] The term "solid phase" describes a non-aqueous matrix to which the
antibody of the present invention can adhere. Examples of solid phases
encompassed herein include those formed partially or entirely of glass
(e.g., controlled pore glass), polysaccharides (e.g., agarose),
polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain
embodiments, depending on the context, the solid phase can comprise the
well of an assay plate; in others it is a purification column (e.g., an
affinity chromatography column). This term also includes a discontinuous
solid phase of discrete particles, such as those described in U.S. Pat.
No. 4,275,149.

[0187] "Antibody effector functions" refer to those biological activities
attributable to the Fc region (a native sequence Fc region or amino acid
sequence variant Fc region) of an antibody, and vary with the antibody
isotype. Examples of antibody effector functions include: C1q binding and
complement dependent cytotoxicity; Fc receptor binding;
antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down
regulation of cell surface receptors (e.g., B cell receptors); and B cell
activation. "Reduced or minimized" antibody effector function means that
which is reduced by at least 50% (alternatively 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, 99%) from the wild type or unmodified
antibody. The determination of antibody effector function is readily
determinable and measurable by one of ordinary skill in the art. In a
preferred embodiment, the antibody effector functions of complement
binding, complement dependent cytotoxicity and antibody dependent
cytotoxicity are affected. In some embodiments of the invention, effector
function is eliminated through a mutation in the constant region that
eliminated glycosylation, e.g., "effector-less mutation." In one aspect,
the effector-less mutation is an N297A or DANA mutation (D265A+N297A) in
the CH2 region. Shields et al., J. Biol. Chem. 276 (9): 6591-6604 (2001).
Alternatively, additional mutations resulting in reduced or eliminated
effector function include: K322A and L234A/L235A (LALA). Alternatively,
effector function can be reduced or eliminated through production
techniques, such as expression in host cells that do not glycosylate
(e.g., E. coli.) or in which result in an altered glycolsylation pattern
that is ineffective or less effective at promoting effector function
(e.g., Shinkawa et al., J. Biol. Chem. 278(5): 3466-3473 (2003).

[0188] "Antibody-dependent cell-mediated cytotoxicity" or ADCC refers to a
form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)
present on certain cytotoxic cells (e.g., natural killer (NK) cells,
neutrophils and macrophages) enable these cytotoxic effector cells to
bind specifically to an antigen-bearing target cell and subsequently kill
the target cell with cytotoxins. The antibodies "arm" the cytotoxic cells
and are required for killing of the target cell by this mechanism. The
primary cells for mediating ADCC, NK cells, express FcγRIII only,
whereas monocytes express FcγRI, FcγRII and FcγRIII. Fc
expression on hematopoietic cells is summarized in Table 3 on page 464 of
Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991). To assess ADCC
activity of a molecule of interest, an in vitro ADCC assay, such as that
described in U.S. Pat. No. 5,500,362 or 5,821,337 may be performed.
Useful effector cells for such assays include peripheral blood
mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or
additionally, ADCC activity of the molecule of interest may be assessed
in vivo, e.g., in an animal model such as that disclosed in Clynes et
al., PNAS USA 95:652-656 (1998).

[0189] Unless indicated otherwise herein, the numbering of the residues in
an immunoglobulin heavy chain is that of the EU index as in Kabat et al.,
supra. The "EU index as in Kabat" refers to the residue numbering of the
human IgG1 EU antibody.

[0190] The term "Fc region" herein is used to define a C-terminal region
of an immunoglobulin heavy chain, including native-sequence Fc regions
and variant Fc regions. Although the boundaries of the Fc region of an
immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc
region is usually defined to stretch from an amino acid residue at
position Cys226, or from Pro230, to the carboxyl-terminus thereof. The
C-terminal lysine (residue 447 according to the EU numbering system) of
the Fc region may be removed, for example, during production or
purification of the antibody, or by recombinantly engineering the nucleic
acid encoding a heavy chain of the antibody. Accordingly, a composition
of intact antibodies may comprise antibody populations with all K447
residues removed, antibody populations with no K447 residues removed, and
antibody populations having a mixture of antibodies with and without the
K447 residue. Suitable native-sequence Fc regions for use in the
antibodies of the invention include human IgG1, IgG2 (IgG2A, IgG2B), IgG3
and IgG4.

[0191] "Fc receptor" or "FcR" describes a receptor that binds to the Fc
region of an antibody. The preferred FcR is a native sequence human FcR.
Moreover, a preferred FcR is one which binds an IgG antibody (a gamma
receptor) and includes receptors of the FcγRI, FcγRII, and
FcγRIII subclasses, including allelic variants and alternatively
spliced forms of these receptors, FcγRII receptors include
FcγRIIA (an "activating receptor") and FcγRIIB (an
"inhibiting receptor"), which have similar amino acid sequences that
differ primarily in the cytoplasmic domains thereof. Activating receptor
FcγRIIA contains an immunoreceptor tyrosine-based activation motif
(ITAM) in its cytoplasmic domain Inhibiting receptor FcγRIIB
contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its
cytoplasmic domain. (see M. Daeron, Annu. Rev. Immunol. 15:203-234
(1997). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:
457-92 (1991); Capel et al., Immunomethods 4: 25-34 (1994); and de Haas
et al., J. Lab. Clin. Med. 126: 330-41 (1995). Other FcRs, including
those to be identified in the future, are encompassed by the term "FcR"
herein.

[0194] "Complement dependent cytotoxicity" or "CDC" refers to the lysis of
a target cell in the presence of complement. Activation of the classical
complement pathway is initiated by the binding of the first component of
the complement system (C1q) to antibodies (of the appropriate subclass)
which are bound to their cognate antigen. To assess complement
activation, a CDC assay, e.g., as described in Gazzano-Santoro et al., J.
Immunol. Methods 202: 163 (1996), may be performed. Antibody variants
with altered Fc region amino acid sequences and increased or decreased
C1q binding capability are described in U.S. Pat. No. 6,194,551B1 and
WO99/51642. The contents of those patent publications are specifically
incorporated herein by reference. See, also, Idusogie et al. J. Immunol.
164: 4178-4184 (2000).

[0195] The N-glycosylation site in IgG is at Asn297 in the CH2 domain. The
present invention also provides compositions of an antigen-binding,
humanized antibody having an Fc region with reduced or no effector
function. One manner in which this can be accomplished is an A297N
substitution, which has previously been shown to abolish complement
binding and effector function ("effector-less Fc mutant") in an anti-CD20
antibody. Idusgie et al., supra. As a result of this mutation, the
production of anti-PD-L1 antibodies of the present inventions containing
this Fc mutation in mammalian cells such as CHO will not have any
glycosylation and, which in turn results in reduced or minimal effector
function. Alternatively, antibody effector function may be eliminated
without CH2 substitution by expression in non-mammalian cells such as E.
Coli.

[0196] "Binding affinity" generally refers to the strength of the sum
total of non-covalent interactions between a single binding site of a
molecule (e.g., an antibody) and its binding partner (e.g., an antigen).
Unless indicated otherwise, as used herein, "binding affinity" refers to
intrinsic binding affinity that reflects a 1:1 interaction between
members of a binding pair (e.g., antibody and antigen). The affinity of a
molecule X for its partner Y can generally be represented by the
dissociation constant (Kd). Affinity can be measured by common methods
known in the art, including those described herein. Low-affinity
antibodies generally bind antigen slowly and tend to dissociate readily,
whereas high-affinity antibodies generally bind antigen faster and tend
to remain bound longer. A variety of methods of measuring binding
affinity are known in the art, any of which can be used for purposes of
the present invention. Specific illustrative and exemplary embodiments
for measuring binding affinity are described in the following.

[0197] The "Kd" or "Kd value" according to this invention is in one
embodiment measured by a radiolabeled antigen binding assay (RIA)
performed with the Fab version of the antibody and antigen molecule as
described by the following assay that measures solution binding affinity
of Fabs for antigen by equilibrating Fab with a minimal concentration of
(125I)-labeled antigen in the presence of a titration series of
unlabeled antigen, then capturing bound antigen with an anti-Fab
antibody-coated plate (Chen, et al., (1999) J. Mol Biol 293:865-881). To
establish conditions for the assay, microtiter plates (Dynex) are coated
overnight with 5 ug/ml of a capturing anti-Fab antibody (Cappel Labs) in
50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v)
bovine serum albumin in PBS for two to five hours at room temperature
(approximately 23° C.). In a non-adsorbant plate (Nunc #269620),
100 pM or 26 pM [125I]-antigen are mixed with serial dilutions of a
Fab of interest (consistent with assessement of an anti-VEGF antibody,
Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599). The Fab of
interest is then incubated overnight; however, the incubation may
continue for a longer period (e.g., 65 hours) to insure that equilibrium
is reached. Thereafter, the mixtures are transferred to the capture plate
for incubation at room temperature for one hour. The solution is then
removed and the plate washed eight times with 0.1% Tween-20 in PBS. When
the plates have dried, 150 ul/well of scintillant (MicroScint-20;
Packard) is added, and the plates are counted on a Topcount gamma counter
(Packard) for ten minutes. Concentrations of each Fab that give less than
or equal to 20% of maximal binding are chosen for use in competitive
binding assays.

[0198] According to another embodiment, the Kd is measured by using
surface-plasmon resonance assays using a BIACORE®-2000 or a
BIACORE®-3000 instrument (BIAcore, Inc., Piscataway, N.J.) at
25° C. with immobilized antigen CM5 chips at ˜10 response
units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5,
BIAcore Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and
N-hydroxysuccinimide (NHS) according to the supplier's instructions.
Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml
(˜0.2 μM) before injection at a flow rate of 5 μL/minute to
achieve approximately 10 response units (RU) of coupled protein.
Following the injection of antigen, 1 M ethanolamine is injected to block
unreacted groups. For kinetics measurements, two-fold serial dilutions of
Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% TWEEN 20®
surfactant (PBST) at 25° C. at a flow rate of approximately 25
μL/min. Association rates (kon) and dissociation rates
(koff) are calculated using a simple one-to-one Langmuir binding
model (BIAcore® Evaluation Software version 3.2) by simultaneously
fitting the association and dissociation sensorgrams. The equilibrium
dissociation constant (Kd) is calculated as the ratio koff/kon.
See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate
exceeds 106M-1 s-1 by the surface-plasmon resonance assay
above, then the on-rate can be determined by using a fluorescent
quenching technique that measures the increase or decrease in
fluorescence-emission intensity (excitation=295 nm; emission=340 nm, 16
nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab
form) in PBS, pH 7.2, in the presence of increasing concentrations of
antigen as measured in a spectrometer, such as a stop-flow-equipped
spectrophotometer (Aviv Instruments) or a 8000-series SLM-AMINCO®
spectrophotometer (ThermoSpectronic) with a stirred cuvette.

[0199] An "on-rate," "rate of association," "association rate," or
"kon" according to this invention can also be determined as
described above using a BIACORE®-2000 or a BIACORE®-3000 system
(BIAcore, Inc., Piscataway, N.J.) at 25° C. with immobilized
antigen CM5 chips at about 10 response units (RU). Briefly,
carboxymethylated dextran biosensor ships (CM5, BIAcore Inc.) are
activated with N-ethyl-N'-(3-dimethylamino propyl)-carbodiimide
hydrochloride (ECD) and N-hydroxysuccinimide (NHS) according to the
supplier's instructions. Antigen is diluted with 10 mM sodium acetate, ph
4.8, into 5 mg/ml (≈0.2 mM) before injection at a flow rate of 5
ml/min. to achieve approximately 10 response units (RU) of coupled
protein. Following the injection of antigen, 1M ethanolamine is added to
block unreacted groups. For kinetics measurements, two-fold serial
dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% Tween
20 (PBST) at 25° C. at a flow rate of approximately 25 ul/min.
Association rates (kon) and dissociation rates (koff) are
calculated using a simple one-to-one Langmuir binding model (BIAcore
Evaluation Software version 3.2) by simultaneous fitting the association
and dissociation sensorgram. The equilibrium dissociation constant (Kd)
was calculated as the ratio koff/kon. See, e.g., Chen, Y., et
al., (1999) J. Mol Biol 293:865-881. However, if the on-rate exceeds
106 M-1 S-1 by the surface plasmon resonance assay above,
then the on-rate is preferably determined by using a fluorescent
quenching technique that measures the increase or decrease in
fluorescence emission intensity (excitation=295 nm; emission=340 nm, 16
nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab
form) in PBS, pH 7.2, in the presence of increasing concentrations of
antigen as measured in a spectrometer, such as a stop-flow equipped
spectrophometer (Aviv Instruments) or a 8000-series SLM-Aminco
spectrophotometer (ThermoSpectronic) with a stirred cuvette.

[0200] The phrase "substantially reduced," or "substantially different,"
as used herein, denotes a sufficiently high degree of difference between
two numeric values (generally one associated with a molecule and the
other associated with a reference/comparator molecule) such that one of
skill in the art would consider the difference between the two values to
be of statistical significance within the context of the biological
characteristic measured by said values (e.g., Kd values). The difference
between said two values is, for example, greater than about 10%, greater
than about 20%, greater than about 30%, greater than about 40%, and/or
greater than about 50% as a function of the value for the
reference/comparator molecule.

[0201] The term "substantially similar" or "substantially the same," as
used herein, denotes a sufficiently high degree of similarity between two
numeric values (for example, one associated with an antibody of the
invention and the other associated with a reference/comparator antibody),
such that one of skill in the art would consider the difference between
the two values to be of little or no biological and/or statistical
significance within the context of the biological characteristic measured
by said values (e.g., Kd values). The difference between said two values
is, for example, less than about 50%, less than about 40%, less than
about 30%, less than about 20%, and/or less than about 10% as a function
of the reference/comparator value.

[0202] "Percent (%) amino acid sequence identity" and "homology" with
respect to a peptide, polypeptide or antibody sequence are defined as the
percentage of amino acid residues in a candidate sequence that are
identical with the amino acid residues in the specific peptide or
polypeptide sequence, after aligning the sequences and introducing gaps,
if necessary, to achieve the maximum percent sequence identity, and not
considering any conservative substitutions as part of the sequence
identity. Alignment for purposes of determining percent amino acid
sequence identity can be achieved in various ways that are within the
skill in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN or MEGALIGN® (DNASTAR)
software. Those skilled in the art can determine appropriate parameters
for measuring alignment, including any algorithms needed to achieve
maximal alignment over the full length of the sequences being compared.
For purposes herein, however, % amino acid sequence identity values are
generated using the sequence comparison computer program ALIGN-2,
authored by Genentech, Inc. The source code of ALIGN-2 has been filed
with user documentation in the U.S. Copyright Office, Washington D.C.,
20559, where it is registered under U.S. Copyright Registration No.
TXU510087. The ALIGN-2 program is publicly available through Genentech,
Inc., South San Francisco, Calif. The ALIGN-2 program should be compiled
for use on a UNIX operating system, preferably digital UNIX V4.0D. All
sequence comparison parameters are set by the ALIGN-2 program and do not
vary.

[0203] In situations where ALIGN-2 is employed for amino acid sequence
comparisons, the % amino acid sequence identity of a given amino acid
sequence A to, with, or against a given amino acid sequence B (which can
alternatively be phrased as a given amino acid sequence A that has or
comprises a certain % amino acid sequence identity to, with, or against a
given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matches
by the sequence alignment program ALIGN-2 in that program's alignment of
A and B, and where Y is the total number of amino acid residues in B. It
will be appreciated that where the length of amino acid sequence A is not
equal to the length of amino acid sequence B, the % amino acid sequence
identity of A to B will not equal the % amino acid sequence identity of B
to A.

[0204] Unless specifically stated otherwise, all % amino acid sequence
identity values used herein are obtained as described in the immediately
preceding paragraph using the ALIGN-2 computer program.

[0205] An "isolated" nucleic acid molecule encoding the antibodies herein
is a nucleic acid molecule that is identified and separated from at least
one contaminant nucleic acid molecule with which it is ordinarily
associated in the environment in which it was produced. Preferably, the
isolated nucleic acid is free of association with all components
associated with the production environment. The isolated nucleic acid
molecules encoding the polypeptides and antibodies herein is in a form
other than in the form or setting in which it is found in nature.
Isolated nucleic acid molecules therefore are distinguished from nucleic
acid encoding the polypeptides and antibodies herein existing naturally
in cells.

[0206] The term "control sequences" refers to DNA sequences necessary for
the expression of an operably linked coding sequence in a particular host
organism. The control sequences that are suitable for prokaryotes, for
example, include a promoter, optionally an operator sequence, and a
ribosome binding site. Eukaryotic cells are known to utilize promoters,
polyadenylation signals, and enhancers.

[0207] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For example,
DNA for a presequence or secretory leader is operably linked to DNA for a
polypeptide if it is expressed as a preprotein that participates in the
secretion of the polypeptide; a promoter or enhancer is operably linked
to a coding sequence if it affects the transcription of the sequence; or
a ribosome binding site is operably linked to a coding sequence if it is
positioned so as to facilitate translation. Generally, "operably linked"
means that the DNA sequences being linked are contiguous, and, in the
case of a secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not exist, the
synthetic oligonucleotide adaptors or linkers are used in accordance with
conventional practice.

[0208] The term "epitope tagged" when used herein refers to a chimeric
polypeptide comprising a polypeptide or antibody described herein fused
to a "tag polypeptide". The tag polypeptide has enough residues to
provide an epitope against which an antibody can be made, yet is short
enough such that it does not interfere with activity of the polypeptide
to which it is fused. The tag polypeptide preferably also is fairly
unique so that the antibody does not substantially cross-react with other
epitopes. Suitable tag polypeptides generally have at least six amino
acid residues and usually between about 8 and 50 amino acid residues
(preferably, between about 10 and 20 amino acid residues).

[0209] As used herein, the term "immunoadhesin" designates antibody-like
molecules which combine the binding specificity of a heterologous protein
(an "adhesion") with the effector functions of immunoglobulin constant
domains. Structurally, the immunoadhesins comprise a fusion of an amino
acid sequence with the desired binding specificity which is other than
the antigen recognition and binding site of an antibody (i.e., is
"heterologous"), and an immunoglobulin constant domain sequence. The
adhesin part of an immunoadhesin molecule typically is a contiguous amino
acid sequence comprising at least the binding site of a receptor or a
ligand. The immunoglobulin constant domain sequence in the immunoadhesin
may be obtained from any immunoglobulin, such as IgG-1, IgG-2 (including
IgG2A and IgG2B), IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and
IgA-2), IgE, IgD or IgM. The Ig fusions preferably include the
substitution of a domain of a polypeptide or antibody described herein in
the place of at least one variable region within an Ig molecule. In a
particularly preferred embodiment, the immunoglobulin fusion includes the
hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgG1
molecule. For the production of immunoglobulin fusions see also U.S. Pat.
No. 5,428,130 issued Jun. 27, 1995. For example, useful immunoadhesins as
second medicaments useful for combination therapy herein include
polypeptides that comprise the extracellular or PD-1 binding portions of
PD-L1 or PD-L2, or vice versa, fused to a constant domain of an
immunoglobulin sequence.

[0210] A "fusion protein" and a "fusion polypeptide" refer to a
polypeptide having two portions covalently linked together, where each of
the portions is a polypeptide having a different properly. The property
may be a biological property, such as activity in vitro or in vivo. The
property may also be simple chemical or physical property, such as
binding to a target molecule, catalysis of a reaction, etc. The two
portions may be linked directly by a single peptide bond or through a
peptide linker will be in reading frame with each other.

[0211] A "stable" formulation is one in which the protein therein
essentially retains its physical and chemical stability and integrity
upon storage. Various analytical techniques for measuring protein
stability are available in the art and are reviewed in Peptide and
Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New
York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90
(1993). Stability can be measured at a selected temperature for a
selected time period. For rapid screening, the formulation may be kept at
40° C. for 2 weeks to 1 month, at which time stability is
measured. Where the formulation is to be stored at 2-8° C.,
generally the formulation should be stable at 30° C. or 40°
C. for at least 1 month and/or stable at 2-8° C. for at least 2
years. Where the formulation is to be stored at 30° C., generally
the formulation should be stable for at least 2 years at 30° C.
and/or stable at 40° C. for at least 6 months. For example, the
extent of aggregation during storage can be used as an indicator of
protein stability. Thus, a "stable" formulation may be one wherein less
than about 10% and preferably less than about 5% of the protein are
present as an aggregate in the formulation. In other embodiments, any
increase in aggregate formation during storage of the formulation can be
determined.

[0212] A "reconstituted" formulation is one which has been prepared by
dissolving a lyophilized protein or antibody formulation in a diluent
such that the protein is dispersed throughout. The reconstituted
formulation is suitable for administration (e.g. subscutaneous
administration) to a patient to be treated with the protein of interest
and, in certain embodiments of the invention, may be one which is
suitable for parenteral or intravenous administration.

[0213] An "isotonic" formulation is one which has essentially the same
osmotic pressure as human blood. Isotonic formulations will generally
have an osmotic pressure from about 250 to 350 mOsm. The term "hypotonic"
describes a formulation with an osmotic pressure below that of human
blood. Correspondingly, the term "hypertonic" is used to describe a
formulation with an osmotic pressure above that of human blood.
Isotonicity can be measured using a vapor pressure or ice-freezing type
osmometer, for example. The formulations of the present invention are
hypertonic as a result of the addition of salt and/or buffer.

[0214] "Carriers" as used herein include pharmaceutically acceptable
carriers, excipients, or stabilizers that are nontoxic to the cell or
mammal being exposed thereto at the dosages and concentrations employed.
Often the physiologically acceptable carrier is an aqueous pH buffered
solution. Examples of physiologically acceptable carriers include buffers
such as phosphate, citrate, and other organic acids; antioxidants
including ascorbic acid; low molecular weight (less than about 10
residues) polypeptide; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino
acids such as glycine, glutamine, asparagine, arginine or lysine;
monosaccharides, disaccharides, and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA; sugar
alcohols such as mannitol or sorbitol; salt-forming counterions such as
sodium; and/or nonionic surfactants such as TWEEN®, polyethylene
glycol (PEG), and PLURONICS®

[0215] A "package insert" refers to instructions customarily included in
commercial packages of medicaments that contain information about the
indications customarily included in commercial packages of medicaments
that contain information about the indications, usage, dosage,
administration, contraindications, other medicaments to be combined with
the packaged product, and/or warnings concerning the use of such
medicaments, etc.

[0218] "Pharmaceutically acceptable" buffers and salts include those
derived from both acid and base addition salts of the above indicated
acids and bases. Specific buffers and/or salts include histidine,
succinate and acetate.

[0219] A "pharmaceutically acceptable sugar" is a molecule which, when
combined with a protein of interest, significantly prevents or reduces
chemical and/or physical instability of the protein upon storage. When
the formulation is intended to be lyophilized and then reconstituted,
"pharmaceutically acceptable sugars" may also be known as a
"lyoprotectant". Exemplary sugars and their corresponding sugar alcohols
include: an amino acid such as monosodium glutamate or histidine; a
methylamine such as betaine; a lyotropic salt such as magnesium sulfate;
a polyol such as trihydric or higher molecular weight sugar alcohols,
e.g. glycerin, dextran, erythritol, glycerol, arabitol, xylitol,
sorbitol, and mannitol; propylene glycol; polyethylene glycol;
PLURONICS®; and combinations thereof. Additional exemplary
lyoprotectants include glycerin and gelatin, and the sugars mellibiose,
melezitose, raffinose, mannotriose and stachyose. Examples of reducing
sugars include glucose, maltose, lactose, maltulose, iso-maltulose and
lactulose. Examples of non-reducing sugars include non-reducing
glycosides of polyhydroxy compounds selected from sugar alcohols and
other straight chain polyalcohols. Preferred sugar alcohols are
monoglycosides, especially those compounds obtained by reduction of
disaccharides such as lactose, maltose, lactulose and maltulose. The
glycosidic side group can be either glucosidic or galactosidic.
Additional examples of sugar alcohols are glucitol, maltitol, lactitol
and iso-maltulose. The preferred pharmaceutically-acceptable sugars are
the non-reducing sugars trehalose or sucrose. Pharmaceutically acceptable
sugars are added to the formulation in a "protecting amount" (e.g.
pre-lyophilization) which means that the protein essentially retains its
physical and chemical stability and integrity during storage (e.g., after
reconstitution and storage).

[0220] The "diluent" of interest herein is one which is pharmaceutically
acceptable (safe and non-toxic for administration to a human) and is
useful for the preparation of a liquid formulation, such as a formulation
reconstituted after lyophilization. Exemplary diluents include sterile
water, bacteriostatic water for injection (BWFI), a pH buffered solution
(e.g. phosphate-buffered saline), sterile saline solution, Ringer's
solution or dextrose solution. In an alternative embodiment, diluents can
include aqueous solutions of salts and/or buffers.

[0221] A "preservative" is a compound which can be added to the
formulations herein to reduce bacterial activity. The addition of a
preservative may, for example, facilitate the production of a multi-use
(multiple-dose) formulation. Examples of potential preservatives include
octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride,
benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides
in which the alkyl groups are long-chain compounds), and benzethonium
chloride. Other types of preservatives include aromatic alcohols such as
phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl
paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol.
The most preferred preservative herein is benzyl alcohol.

[0222] "Treatment" refers to clinical intervention designed to alter the
natural course of the individual or cell being treated, and can be
performed either for prophylaxis or during the course of clinical
pathology. Desirable effects of treatment include preventing occurrence
or recurrence of disease, preventing metastasis, decreasing the rate of
disease progression, ameliorating or palliating the disease state, and
remission or improved prognosis. In some embodiments, antibodies of the
invention are used to delay development of a disease or disorder. A
subject is successfully "treated", for example, using the apoptotic
anti-PD-L1 antibodies of the invention if one or more symptoms associated
with a T-cell dysfunctional disorder is mitigated.

[0223] An "effective amount" refers to at least an amount effective, at
dosages and for periods of time necessary, to achieve the desired or
indicated effect, including a therapeutic or prophylactic result. For
example, an effective amount of the anti-PD-L1 antibodies of the present
invention is at least the minimum concentration that results in
inhibition of signaling from PD-L1, either through PD-1 on T-cells or
B7.1 on other APCs or both.

[0224] A "therapeutically effective amount" is at least the minimum
concentration required to effect a measurable improvement or prevention
of a particular disorder. A therapeutically effective amount herein may
vary according to factors such as the disease state, age, sex, and weight
of the patient, and the ability of the antibody to elicit a desired
response in the individual. A therapeutically effective amount is also
one in which any toxic or detrimental effects of the antibody are
outweighed by the therapeutically beneficial effects. For example, a
therapeutically effective amount of the anti-PD-L1 antibodies of the
present invention is at least the minimum concentration that results in
inhibition of at least one symptom of a T cell dysfunctional disorder.

[0225] A "prophylactically effective amount" refers to an amount
effective, at the dosages and for periods of time necessary, to achieve
the desired prophylactic result. For example, a prophylactically
effective amount of the anti-PD-L1 antibodies of the present invention is
at least the minimum concentration that prevents or attenuates the
development of at least one symptom of a T cell dysfunctional disorder.

[0226] "Chronic" administration refers to administration of the
medicament(s) in a continuous as opposed to acute mode, so as to main the
initial therapeutic effect (activity) for an extended period of time.
"Intermittent" administration is treatment that is not consecutively done
without interruption, but rather is cyclic in nature.

[0227] "Mammal" for purposes of treatment refers to any animal classified
as a mammal, including humans, domestic and farm animals, and zoo,
sports, or pet animals, such as dogs, horses, rabbits, cattle, pigs,
hamsters, gerbils, mice, ferrets, rats, cats, etc. Preferably, the mammal
is human.

[0228] The term "pharmaceutical formulation" refers to a preparation that
is in such form as to permit the biological activity of the active
ingredient to be effective, and that contains no additional components
that are unacceptably toxic to a subject to which the formulation would
be administered. Such formulations are sterile.

[0229] A "sterile" formulation is aseptic or free from all living
microorganisms and their spores.

[0230] The term "about" as used herein refers to the usual error range for
the respective value readily known to the skilled person in this
technical field.

[0232] The term "cytotoxic agent" as used herein refers to a substance
that inhibits or prevents the function of cells and/or causes destruction
of cells. The term includes radioactive isotopes (e.g. At211,
I131, I125, Y90, Re186, Re188, Sm153,
Bi212, P32 and radioactive isotopes of Lu), and toxins such as
small-molecule toxins or enzymatically active toxins of bacterial,
fungal, plant or animal origin, or fragments thereof.

[0234] Also included in this definition are anti-hormonal agents that act
to regulate, reduce, block, or inhibit the effects of hormones that can
promote the growth of cancer, and are often in the form of systemic, or
whole-body treatment. They may be hormones themselves. Examples include
anti-estrogens and selective estrogen receptor modulators (SERMs),
including, for example, tamoxifen (including NOLVADEX® tamoxifen),
raloxifene (EVISTA®), droloxifene, 4-hydroxytamoxifen, trioxifene,
keoxifene, LY117018, onapristone, and toremifene (FARESTON®);
anti-progesterones; estrogen receptor down-regulators (ERDs); estrogen
receptor antagonists such as fulvestrant (FASLODEX®); agents that
function to suppress or shut down the ovaries, for example, leutinizing
hormone-releasing hormone (LHRH) agonists such as leuprolide acetate
(LUPRON® and ELIGARD®), goserelin acetate, buserelin acetate and
tripterelin; anti-androgens such as flutamide, nilutamide and
bicalutamide; and aromatase inhibitors that inhibit the enzyme aromatase,
which regulates estrogen production in the adrenal glands, such as, for
example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate
(MEGASE®), exemestane (AROMASIN®), formestanie, fadrozole,
vorozole (RIVISOR®), letrozole (FEMARA®), and anastrozole
(ARIMIDEX®). In addition, such definition of chemotherapeutic agents
includes bisphosphonates such as clodronate (for example, BONEFOS® or
OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic
acid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate
(AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®);
as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog);
anti-sense oligonucleotides, particularly those that inhibit expression
of genes in signaling pathways implicated in abherant cell proliferation,
such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor
receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene
therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN®
vaccine, and VAXID® vaccine; topoisomerase 1 inhibitor (e.g.,
LURTOTECAN®); an anti-estrogen such as fulvestrant; a Kit inhibitor
such as imatinib or EXEL-0862 (a tyrosine kinase inhibitor); EGFR
inhibitor such as erlotinib or cetuximab; an anti-VEGF inhibitor such as
bevacizumab; arinotecan; rmRH (e.g., ABARELIX®); lapatinib and
lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosine kinase
small-molecule inhibitor also known as GW572016); 17AAG (geldanamycin
derivative that is a heat shock protein (Hsp) 90 poison), and
pharmaceutically acceptable salts, acids or derivatives of any of the
above.

[0235] A "growth-inhibitory agent" refers to a compound or composition
that inhibits growth of a cell, which growth depends on receptor
activation either in vitro or in vivo. Thus, the growth-inhibitory agent
includes one that significantly reduces the percentage of
receptor-dependent cells in S phase. Examples of growth-inhibitory agents
include agents that block cell-cycle progression (at a place other than S
phase), such as agents that induce G1 arrest and M-phase arrest.
Classical M-phase blockers include the vincas and vinca alkaloids
(vincristine and vinblastine), taxanes, and topoisomerase II inhibitors
such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.
Those agents that arrest G1 also spill over into S-phase arrest, for
example, DNA alkylating agents such as tamoxifen, prednisone,
dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil,
and ara-C. Further information can be found in The Molecular Basis of
Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle
regulation, oncogenes, and antineoplastic drugs" by Murakami et al. (WB
Saunders: Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel
and docetaxel) are anticancer drugs both derived from the yew tree.
Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the European
yew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers
Squibb).

[0236] The term "cytokine" is a generic term for proteins released by one
cell population that act on another cell as intercellular mediators.
Examples of such cytokines are lymphokines, monokines; interleukins (ILs)
such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,
IL-9, IL-11, IL-12, IL-13, IL-15 . . . IL-35, including PROLEUKIN®
rIL-2; a tumor-necrosis factor such as TNF-α or TNF-β; and
other polypeptide factors including LIF and kit ligand (KL), while the
term "interleukin" has now essentially become a synonym for cytokine. As
used herein, the term cytokine includes proteins from natural sources or
from recombinant cell culture and biologically active equivalents of the
native-sequence cytokines, including synthetically produced
small-molecule entities and pharmaceutically acceptable derivatives and
salts thereof. Cytokines can be classified on the proximal location of
the intended target, wherein autocrine refers to action on the same cell
from which it is secreted, paracrine refers to action restricted to the
immediate vicinity into which the cytokine is secreted, and endocrine
refers to action in distant regions of the body. Immune cytokines can
also be classified by whether they enhance a type I response, (e.g.,
IFN-γ, TGF-β etc), which favor cellular immunity or a type II
response (IL-4, IL-10, IL-13, etc.), which favor antibody or humoral
immunity. Immune cytokines play roles in co-stimulation, maturation,
proliferation, activation, inflammation, growth, differentiation,
cytokines production and secretion, survival of various immune cells.

[0240] Phage(mid) display (also referred to herein as phage display) can
be used as a convenient and fast method for generating and screening many
different potential variant antibodies in a library generated by sequence
randomization. However, other methods for making and screening altered
antibodies are available to the skilled person.

[0241] Phage(mid) display (also referred to herein as phage display in
some contexts) can be used as a convenient and fast method for generating
and screening many different potential variant antibodies in a library
generated by sequence randomization. However, other methods for making
and screening altered antibodies are available to the skilled person.

[0242] Phage(mid) display technology has provided a powerful tool for
generating and selecting novel proteins which bind to a ligand, such as
an antigen. Using the techniques of phage(mid) display allows the
generation of large libraries of protein variants which can be rapidly
sorted for those sequences that bind to a target molecule with high
affinity. Nucleic acids encoding variant polypeptides are generally fused
to a nucleic acid sequence encoding a viral coat protein, such as the
gene III protein or the gene VIII protein. Monovalent phagemid display
systems where the nucleic acid sequence encoding the protein or
polypeptide is fused to a nucleic acid sequence encoding a portion of the
gene III protein have been developed. (Bass, S., Proteins, 8:309 (1990);
Lowman and Wells, Methods: A Companion to Methods in Enzymology, 3:205
(1991)). In a monovalent phagemid display system, the gene fusion is
expressed at low levels and wild type gene III proteins are also
expressed so that infectivity of the particles is retained. Methods of
generating peptide libraries and screening those libraries have been
disclosed in many patents (e.g. U.S. Pat. No. 5,723,286, U.S. Pat. No.
5,432,018, U.S. Pat. No. 5,580,717, U.S. Pat. No. 5,427,908 and U.S. Pat.
No. 5,498,530).

[0243] Libraries of antibodies or antigen binding polypeptides have been
prepared in a number of ways including by altering a single gene by
inserting random DNA sequences or by cloning a family of related genes.
Methods for displaying antibodies or antigen binding fragments using
phage(mid) display have been described in U.S. Pat. Nos. 5,750,373,
5,733,743, 5,837,242, 5,969,108, 6,172,197, 5,580,717, and 5,658,727. The
library is then screened for expression of antibodies or antigen binding
proteins with the desired characteristics.

[0244] Methods of substituting an amino acid of choice into a template
nucleic acid are well established in the art, some of which are described
herein. For example, hypervariable region residues can be substituted
using the Kunkel method. See, e.g., Kunkel et al., Methods Enzymol.
154:367-382 (1987).

[0245] The sequence of oligonucleotides includes one or more of the
designed codon sets for the hypervariable region residues to be altered.
A codon set is a set of different nucleotide triplet sequences used to
encode desired variant amino acids. Codon sets can be represented using
symbols to designate particular nucleotides or equimolar mixtures of
nucleotides as shown in below according to the IUB code.

TABLE-US-00016
IUB CODES
G (Guanine) Y (C or T) H (A or C or T)
A (Adenine) M (A or C) B (C or G or T)
T (Thymine) K (G or T) V (A or C or G)
C (Cytosine) S (C or G) D (A or G or T)
R (A or G) W (A or T) N (A or C or G or T)
For example, in the codon set DVK, D can be nucleotides A or G or T; V can
be A or G or C; and K can be G or T. This codon set can present 18
different codons and can encode amino acids Ala, Trp, Tyr, Lys, Thr, Asn,
Lys, Ser, Arg, Asp, Glu, Gly, and Cys.

[0246] Oligonucleotide or primer sets can be synthesized using standard
methods. A set of oligonucleotides can be synthesized, for example, by
solid phase synthesis, containing sequences that represent all possible
combinations of nucleotide triplets provided by the codon set and that
will encode the desired group of amino acids. Synthesis of
oligonucleotides with selected nucleotide "degeneracy" at certain
positions is well known in that art. Such sets of nucleotides having
certain codon sets can be synthesized using commercial nucleic acid
synthesizers (available from, for example, Applied Biosystems, Foster
City, Calif.), or can be obtained commercially (for example, from Life
Technologies, Rockville, Md.). Therefore, a set of oligonucleotides
synthesized having a particular codon set will typically include a
plurality of oligonucleotides with different sequences, the differences
established by the codon set within the overall sequence.
Oligonucleotides, as used according to the invention, have sequences that
allow for hybridization to a variable domain nucleic acid template and
also can include restriction enzyme sites for cloning purposes.

[0247] In one method, nucleic acid sequences encoding variant amino acids
can be created by oligonucleotide-mediated mutagenesis. This technique is
well known in the art as described by Zoller et al. Nucleic Acids Res.
10:6487-6504 (1987). Briefly, nucleic acid sequences encoding variant
amino acids are created by hybridizing an oligonucleotide set encoding
the desired codon sets to a DNA template, where the template is the
single-stranded form of the plasmid containing a variable region nucleic
acid template sequence. After hybridization, DNA polymerase is used to
synthesize an entire second complementary strand of the template that
will thus incorporate the oligonucleotide primer, and will contain the
codon sets as provided by the oligonucleotide set.

[0248] Generally, oligonucleotides of at least 25 nucleotides in length
are used. An optimal oligonucleotide will have 12 to 15 nucleotides that
are completely complementary to the template on either side of the
nucleotide(s) coding for the mutation(s). This ensures that the
oligonucleotide will hybridize properly to the single-stranded DNA
template molecule. The oligonucleotides are readily synthesized using
techniques known in the art such as that described by Crea et al., Proc.
Nat'l. Acad. Sci. USA, 75:5765 (1978).

[0249] The DNA template is generated by those vectors that are either
derived from bacteriophage M13 vectors (the commercially available
M13mp18 and M13mp19 vectors are suitable), or those vectors that contain
a single-stranded phage origin of replication as described by Viera et
al., Meth. Enzymol., 153:3 (1987). Thus, the DNA that is to be mutated
can be inserted into one of these vectors in order to generate a
single-stranded template. Production of the single-stranded template is
described in sections 4.21-4.41 of Sambrook et al., above.

[0250] To alter the native DNA sequence, the oligonucleotide is hybridized
to the single stranded template under suitable hybridization conditions.
A DNA polymerizing enzyme, usually T7 DNA polymerase or the Klenow
fragment of DNA polymerase I, is then added to synthesize the
complementary strand of the template using the oligonucleotide as a
primer for synthesis. A heteroduplex molecule is thus formed such that
one strand of DNA encodes the mutated form of gene 1, and the other
strand (the original template) encodes the native, unaltered sequence of
gene 1. This heteroduplex molecule is then transformed into a suitable
host cell, usually a prokaryote such as E. coli JM101. After growing the
cells, they are plated onto agarose plates and screened using the
oligonucleotide primer radiolabelled with a 32-Phosphate to identify
the bacterial colonies that contain the mutated DNA.

[0251] The method described immediately above may be modified such that a
homoduplex molecule is created wherein both strands of the plasmid
contain the mutation(s). The modifications are as follows: The single
stranded oligonucleotide is annealed to the single-stranded template as
described above. A mixture of three deoxyribonucleotides,
deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), and
deoxyribothymidine (dTT), is combined with a modified
thiodeoxyribocytosine called dCTP-(aS) (which can be obtained from
Amersham). This mixture is added to the template-oligonucleotide complex.
Upon addition of DNA polymerase to this mixture, a strand of DNA
identical to the template except for the mutated bases is generated. In
addition, this new strand of DNA will contain dCTP-(aS) instead of dCTP,
which serves to protect it from restriction endonuclease digestion. After
the template strand of the double-stranded heteroduplex is nicked with an
appropriate restriction enzyme, the template strand can be digested with
ExoIII nuclease or another appropriate nuclease to cut at other than the
region that contains the site(s) to be mutagenized. The reaction is then
stopped to leave a molecule that is only partially single-stranded. A
complete double-stranded DNA homoduplex is then formed using DNA
polymerase in the presence of all four deoxyribonucleotide triphosphates,
ATP, and DNA ligase. This homoduplex molecule can then be transformed
into a suitable host cell.

[0252] As indicated previously, the sequence of the oligonucleotide set is
of sufficient length to hybridize to the template nucleic acid and may
also, but does not necessarily, contain restriction sites. The DNA
template can be generated by those vectors that are either derived from
bacteriophage M13 vectors or vectors that contain a single-stranded phage
origin of replication as described by Viera et al. Meth. Enzymol., 153:3
(1987). Thus, the DNA that is to be mutated must be inserted into one of
these vectors in order to generate a single-stranded template. Production
of the single-stranded template is described in sections 4.21-4.41 of
Sambrook et al., supra.

[0253] According to another method, a library can be generated by
providing upstream and downstream oligonucleotide sets, each set having a
plurality of oligonucleotides with different sequences, the different
sequences established by the codon sets provided within the sequence of
the oligonucleotides. The upstream and downstream oligonucleotide sets,
along with a variable domain template nucleic acid sequence, can be used
in a polymerase chain reaction to generate a "library" of PCR products.
The PCR products can be referred to as "nucleic acid cassettes", as they
can be fused with other related or unrelated nucleic acid sequences, for
example, viral coat proteins and dimerization domains, using established
molecular biology techniques.

[0254] The sequence of the PCR primers includes one or more of the
designed codon sets for the solvent accessible and highly diverse
positions in a hypervariable region. As described above, a codon set is a
set of different nucleotide triplet sequences used to encode desired
variant amino acids. Antibody selectants that meet the desired criteria,
as selected through appropriate screening/selection steps can be isolated
and cloned using standard recombinant techniques.

[0255] B. Recombinant Preparation

[0256] The invention also provides an isolated nucleic acid encoding
anti-PD-L1 antibodies, vectors and host cells comprising such nucleic
acid, and recombinant techniques for the production of the antibody.

[0257] For recombinant production of the antibody, the nucleic acid
encoding it is isolated and inserted into a replicable vector for further
cloning (amplification of the DNA) or for expression. DNA encoding the
monoclonal antibody is readily isolated and sequenced using conventional
procedures (e.g., by using oligonucleotide probes that are capable of
binding specifically to genes encoding the heavy and light chains of the
antibody). Many vectors are available. The choice of vector depends in
part on the host cell to be used. Generally, preferred host cells are of
either prokaryotic or eukaryotic (generally mammalian) origin.

[0258] 1. Antibody Production in Prokaryotic Cells

[0259] a) Vector Construction

[0260] Polynucleotide sequences encoding polypeptide components of the
antibodies of the invention can be obtained using standard recombinant
techniques. Desired polynucleotide sequences may be isolated and
sequenced from antibody producing cells such as hybridoma cells.
Alternatively, polynucleotides can be synthesized using nucleotide
synthesizer or PCR techniques. Once obtained, sequences encoding the
polypeptides are inserted into a recombinant vector capable of
replicating and expressing heterologous polynucleotides in prokaryotic
hosts. Many vectors that are available and known in the art can be used
for the purpose of the present invention. Selection of an appropriate
vector will depend mainly on the size of the nucleic acids to be inserted
into the vector and the particular host cell to be transformed with the
vector. Each vector contains various components, depending on its
function (amplification or expression of heterologous polynucleotide, or
both) and its compatibility with the particular host cell in which it
resides. The vector components generally include, but are not limited to:
an origin of replication, a selection marker gene, a promoter, a ribosome
binding site (RBS), a signal sequence, the heterologous nucleic acid
insert and a transcription termination sequence.

[0261] In general, plasmid vectors containing replicon and control
sequences which are derived from species compatible with the host cell
are used in connection with these hosts. The vector ordinarily carries a
replication site, as well as marking sequences which are capable of
providing phenotypic selection in transformed cells. For example, E. coli
is typically transformed using pBR322, a plasmid derived from an E. coli
species. pBR322 contains genes encoding ampicillin (Amp) and tetracycline
(Tet) resistance and thus provides easy means for identifying transformed
cells. pBR322, its derivatives, or other microbial plasmids or
bacteriophage may also contain, or be modified to contain, promoters
which can be used by the microbial organism for expression of endogenous
proteins. Examples of pBR322 derivatives used for expression of
particular antibodies are described in detail in Carter et al., U.S. Pat.
No. 5,648,237.

[0262] In addition, phage vectors containing replicon and control
sequences that are compatible with the host microorganism can be used as
transforming vectors in connection with these hosts. For example,
bacteriophage such as GEM®-11 may be utilized in making a recombinant
vector which can be used to transform susceptible host cells such as E.
coli LE392.

[0263] The expression vector of the invention may comprise two or more
promoter-cistron pairs, encoding each of the polypeptide components. A
promoter is an untranslated regulatory sequence located upstream (5') to
a cistron that modulates its expression. Prokaryotic promoters typically
fall into two classes, inducible and constitutive. Inducible promoter is
a promoter that initiates increased levels of transcription of the
cistron under its control in response to changes in the culture
condition, e.g. the presence or absence of a nutrient or a change in
temperature.

[0264] A large number of promoters recognized by a variety of potential
host cells are well known. The selected promoter can be operably linked
to cistron DNA encoding the light or heavy chain by removing the promoter
from the source DNA via restriction enzyme digestion and inserting the
isolated promoter sequence into the vector of the invention. Both the
native promoter sequence and many heterologous promoters may be used to
direct amplification and/or expression of the target genes. In some
embodiments, heterologous promoters are utilized, as they generally
permit greater transcription and higher yields of expressed target gene
as compared to the native target polypeptide promoter.

[0265] Promoters suitable for use with prokaryotic hosts include the PhoA
promoter, the--galactamase and lactose promoter systems, a tryptophan
(trp) promoter system and hybrid promoters such as the tac or the trc
promoter. However, other promoters that are functional in bacteria (such
as other known bacterial or phage promoters) are suitable as well. Their
nucleotide sequences have been published, thereby enabling a skilled
worker operably to ligate them to cistrons encoding the target light and
heavy chains (Siebenlist et al. (1980) Cell 20: 269) using linkers or
adaptors to supply any required restriction sites.

[0266] In one aspect, each cistron within the recombinant vector comprises
a secretion signal sequence component that directs translocation of the
expressed polypeptides across a membrane. In general, the signal sequence
may be a component of the vector, or it may be a part of the target
polypeptide DNA that is inserted into the vector. The signal sequence
selected for the purpose of this invention should be one that is
recognized and processed (i.e. cleaved by a signal peptidase) by the host
cell. For prokaryotic host cells that do not recognize and process the
signal sequences native to the heterologous polypeptides, the signal
sequence is substituted by a prokaryotic signal sequence selected, for
example, from the group consisting of the alkaline phosphatase,
penicillinase, Ipp, or heat-stable enterotoxin II (STII) leaders, LamB,
PhoE, PelB, OmpA and MBP. In one embodiment of the invention, the signal
sequences used in both cistrons of the expression system are STII signal
sequences or variants thereof.

[0267] In another aspect, the production of the immunoglobulins according
to the invention can occur in the cytoplasm of the host cell, and
therefore does not require the presence of secretion signal sequences
within each cistron. In that regard, immunoglobulin light and heavy
chains are expressed, folded and assembled to form functional
immunoglobulins within the cytoplasm. Certain host strains (e.g., the E.
coli trxB.sup.- strains) provide cytoplasm conditions that are favorable
for disulfide bond formation, thereby permitting proper folding and
assembly of expressed protein subunits. Proba and Pluckthun Gene, 159:203
(1995).

[0268] The present invention provides an expression system in which the
quantitative ratio of expressed polypeptide components can be modulated
in order to maximize the yield of secreted and properly assembled
antibodies of the invention. Such modulation is accomplished at least in
part by simultaneously modulating translational strengths for the
polypeptide components. One technique for modulating translational
strength is disclosed in Simmons et al., U.S. Pat. No. 5,840,523. It
utilizes variants of the translational initiation region (TIR) within a
cistron. For a given TIR, a series of amino acid or nucleic acid sequence
variants can be created with a range of translational strengths, thereby
providing a convenient means by which to adjust this factor for the
desired expression level of the specific chain. TIR variants can be
generated by conventional mutagenesis techniques that result in codon
changes which can alter the amino acid sequence, although silent changes
in the nucleotide sequence are preferred. Alterations in the TIR can
include, for example, alterations in the number or spacing of
Shine-Dalgarno sequences, along with alterations in the signal sequence.
One method for generating mutant signal sequences is the generation of a
"codon bank" at the beginning of a coding sequence that does not change
the amino acid sequence of the signal sequence (i.e., the changes are
silent). This can be accomplished by changing the third nucleotide
position of each codon; additionally, some amino acids, such as leucine,
serine, and arginine, have multiple first and second positions that can
add complexity in making the bank. This method of mutagenesis is
described in detail in Yansura et al. (1992) METHODS: A Companion to
Methods in Enzymol. 4:151-158.

[0269] Preferably, a set of vectors is generated with a range of TIR
strengths for each cistron therein. This limited set provides a
comparison of expression levels of each chain as well as the yield of the
desired antibody products under various TIR strength combinations. TIR
strengths can be determined by quantifying the expression level of a
reporter gene as described in detail in Simmons et al. U.S. Pat. No.
5,840,523. Based on the translational strength comparison, the desired
individual TIRs are selected to be combined in the expression vector
constructs of the invention.

[0270] b) Prokaryotic Host Cells.

[0271] Prokaryotic host cells suitable for expressing antibodies of the
invention include Archaebacteria and Eubacteria, such as Gram-negative or
Gram-positive organisms. Examples of useful bacteria include Escherichia
(e.g., E. coli), Bacilli (e.g., B. subtilis), Enterobacteria, Pseudomonas
species (e.g., P. aeruginosa), Salmonella typhimurium, Serratia
marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or
Paracoccus. In one embodiment, gram-negative cells are used. In one
embodiment, E. coli cells are used as hosts for the invention. Examples
of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular
Biology, vol. 2 (Washington, D.C.: American Society for Microbiology,
1987), pp. 1190-1219; ATCC Deposit No. 27,325) and derivatives thereof,
including strain 33D3 having genotype W3110 fhuA ( tonA) ptr3 lac Iq
lacL8 ompT (nmpc-fepE) degP41 kanR (U.S. Pat. No. 5,639,635). Other
strains and derivatives thereof, such as E. coli 294 (ATCC 31,446), E.
coli B, E. coli 1776 (ATCC 31,537) and E. coli RV308 (ATCC 31,608) are
also suitable. These examples are illustrative rather than limiting.
Methods for constructing derivatives of any of the above-mentioned
bacteria having defined genotypes are known in the art and described in,
for example, Bass et al., Proteins, 8:309-314 (1990). It is generally
necessary to select the appropriate bacteria taking into consideration
replicability of the replicon in the cells of a bacterium. For example,
E. coli, Serratia, or Salmonella species can be suitably used as the host
when well known plasmids such as pBR322, pBR325, pACYC177, or pKN410 are
used to supply the replicon.

[0272] Typically the host cell should secrete minimal amounts of
proteolytic enzymes, and additional protease inhibitors may desirably be
incorporated in the cell culture.

[0273] c) Antibody Production

[0274] Host cells are transformed with the above-described expression
vectors and cultured in conventional nutrient media modified as
appropriate for inducing promoters, selecting transformants, or
amplifying the genes encoding the desired sequences. Transformation means
introducing DNA into the prokaryotic host so that the DNA is replicable,
either as an extrachromosomal element or by chromosomal integrant.
Depending on the host cell used, transformation is done using standard
techniques appropriate to such cells. The calcium treatment employing
calcium chloride is generally used for bacterial cells that contain
substantial cell-wall barriers. Another method for transformation employs
polyethylene glycol/DMSO. Yet another technique used is electroporation.

[0275] Prokaryotic cells used to produce the antibodies of the invention
are grown in media known in the art and suitable for culture of the
selected host cells. Examples of suitable media include luria broth (LB)
plus necessary nutrient supplements. In some embodiments, the media also
contains a selection agent, chosen based on the construction of the
expression vector, to selectively permit growth of prokaryotic cells
containing the expression vector. For example, ampicillin is added to
media for growth of cells expressing ampicillin resistant gene.

[0276] Any necessary supplements besides carbon, nitrogen, and inorganic
phosphate sources may also be included at appropriate concentrations
introduced alone or as a mixture with another supplement or medium such
as a complex nitrogen source. Optionally the culture medium may contain
one or more reducing agents selected from the group consisting of
glutathione, cysteine, cystamine, thioglycollate, dithioerythritol and
dithiothreitol.

[0277] The prokaryotic host cells are cultured at suitable temperatures.
For E. coli growth, for example, the preferred temperature ranges from
about 20° C. to about 39° C., more preferably from about
25° C. to about 37° C., even more preferably at about
30° C. The pH of the medium may be any pH ranging from about 5 to
about 9, depending mainly on the host organism. For E. coli, the pH is
preferably from about 6.8 to about 7.4, and more preferably about 7.0.

[0278] If an inducible promoter is used in the expression vector of the
invention, protein expression is induced under conditions suitable for
the activation of the promoter. In one aspect of the invention, PhoA
promoters are used for controlling transcription of the polypeptides.
Accordingly, the transformed host cells are cultured in a
phosphate-limiting medium for induction. Preferably, the
phosphate-limiting medium is the C.R.A.P medium (see, e.g., Simmons et
al., J. Immunol. Methods (2002), 263:133-147). A variety of other
inducers may be used, according to the vector construct employed, as is
known in the art.

[0279] The expressed antibody proteins of the present invention are
secreted into and recovered from the periplasm of the host cells. Protein
recovery typically involves disrupting the microorganism, generally by
such means as osmotic shock, sonication or lysis. Once cells are
disrupted, cell debris or whole cells may be removed by centrifugation or
filtration. The proteins may be further purified, for example, by
affinity resin chromatography. Alternatively, proteins can be transported
into the culture media and isolated therein. Cells may be removed from
the culture and the culture supernatant being filtered and concentrated
for further purification of the proteins produced. The expressed
polypeptides can be further isolated and identified using commonly known
methods such as polyacrylamide gel electrophoresis (PAGE) and Western
blot assay.

[0280] Alternatively, antibody production is conducted in large quantity
by a fermentation process. Various large-scale fed-batch fermentation
procedures are available for production of recombinant proteins.
Large-scale fermentations have at least 1000 liters of capacity,
preferably about 1,000 to 100,000 liters of capacity. These fermentors
use agitator impellers to distribute oxygen and nutrients, especially
glucose (the preferred carbon/energy source). Small scale fermentation
refers generally to fermentation in a fermentor that is no more than
approximately 100 liters in volumetric capacity, and can range from about
1 liter to about 100 liters.

[0281] During the fermentation process, induction of protein expression is
typically initiated after the cells have been grown under suitable
conditions to a desired density, e.g., an OD550 of about 180-220, at
which stage the cells are in the early stationary phase. A variety of
inducers may be used, according to the vector construct employed, as is
known in the art and described above. Cells may be grown for shorter
periods prior to induction. Cells are usually induced for about 12-50
hours, although longer or shorter induction time may be used.

[0282] To improve the production yield and quality of the antibodies of
the invention, various fermentation conditions can be modified. For
example, to improve the proper assembly and folding of the secreted
antibody polypeptides, additional vectors overexpressing chaperone
proteins, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or
FkpA (a peptidylprolyl cis,trans-isomerase with chaperone activity) can
be used to co-transform the host prokaryotic cells. The chaperone
proteins have been demonstrated to facilitate the proper folding and
solubility of heterologous proteins produced in bacterial host cells.
Chen et al. (1999) J Bio Chem 274:19601-19605; Georgiou et al., U.S. Pat.
No. 6,083,715; Georgiou et al., U.S. Pat. No. 6,027,888; Bothmann and
Pluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun
(2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol. Microbiol.
39:199-210.

[0284] E. coli strains deficient for proteolytic enzymes and transformed
with plasmids overexpressing one or more chaperone proteins may be used
as host cells in the expression system encoding the antibodies of the
invention.

[0285] d) Antibody Purification

[0286] The antibody protein produced herein is further purified to obtain
preparations that are substantially homogeneous for further assays and
uses. Standard protein purification methods known in the art can be
employed. The following procedures are exemplary of suitable purification
procedures: fractionation on immunoaffinity or ion-exchange columns,
ethanol precipitation, reverse phase HPLC, chromatography on silica or on
a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE,
ammonium sulfate precipitation, and gel filtration using, for example,
Sephadex G-75.

[0287] In one aspect, Protein A immobilized on a solid phase is used for
immunoaffinity purification of the full length antibody products of the
invention. Protein A is a 41 kD cell wall protein from Staphylococcus
aureas which binds with a high affinity to the Fc region of antibodies.
Lindmark et al (1983) J. Immunol. Meth. 62:1-13. The solid phase to which
Protein A is immobilized is preferably a column comprising a glass or
silica surface, more preferably a controlled pore glass column or a
silicic acid column. In some applications, the column has been coated
with a reagent, such as glycerol, in an attempt to prevent nonspecific
adherence of contaminants. The solid phase is then washed to remove
contaminants non-specifically bound to the solid phase. Finally the
antibody of interest is recovered from the solid phase by elution.

[0288] 2. Antibody Production in Eukaryotic Cells

[0289] For Eukaryotic expression, the vector components generally include,
but are not limited to, one or more of the following, a signal sequence,
an origin of replication, one or more marker genes, and enhancer element,
a promoter, and a transcription termination sequence.

[0290] a) Signal Sequence Component

[0291] A vector for use in a eukaryotic host may also an insert that
encodes a signal sequence or other polypeptide having a specific cleavage
site at the N-terminus of the mature protein or polypeptide. The
heterologous signal sequence selected preferably is one that is
recognized and processed (i.e., cleaved by a signal peptidase) by the
host cell. In mammalian cell expression, mammalian signal sequences as
well as viral secretory leaders, for example, the herpes simplex gD
signal, are available.

[0292] The DNA for such precursor region is ligated in reading frame to
DNA encoding the antibodies of the invention.

[0293] b) Origin of Replication

[0294] Generally, the origin of replication component is not needed for
mammalian expression vectors (the SV40 origin may typically be used only
because it contains the early promoter).

[0297] One example of a selection scheme utilizes a drug to arrest growth
of a host cell. Those cells that are successfully transformed with a
heterologous gene produce a protein conferring drug resistance and thus
survive the selection regimen. Examples of such dominant selection use
the drugs neomycin, mycophenolic acid and hygromycin.

[0298] Another example of suitable selectable markers for mammalian cells
are those that enable the identification of cells competent to take up
nucleic acid encoding the antibodies of the invention, such as DHFR,
thymidine kinase, metallothionein-I and -II, preferably primate
metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.

[0299] For example, cells transformed with the DHFR selection gene are
first identified by culturing all of the transformants in a culture
medium that contains methotrexate (Mtx), a competitive antagonist of
DHFR. An appropriate host cell when wild-type DHFR is employed is the
Chinese hamster ovary (CHO) cell line deficient in DHFR activity (e.g.,
ATCC CRL-9096).

[0300] Alternatively, host cells (particularly wild-type hosts that
contain endogenous DHFR) transformed or co-transformed with the antibody
encoding-DNA sequences, wild-type DHFR protein, and another selectable
marker such as aminoglycoside 3'-phosphotransferase (APH) can be selected
by cell growth in medium containing a selection agent for the selectable
marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin,
or G418. See U.S. Pat. No. 4,965,199.

[0301] d) Promoter Component

[0302] Expression and cloning vectors usually contain a promoter that is
recognized by the host organism and is operably linked to the nucleic
acid encoding the desired antibody sequences. Virtually all eukaryotic
genes have an AT-rich region located approximately 25 to 30 based
upstream from the site where transcription is initiated. Another sequence
found 70 to 80 bases upstream from the start of the transcription of many
genes is a CNCAAT region where N may be any nucleotide. A the 3' end of
most eukaryotic is an AATAAA sequence that may be the signal for addition
of the poly A tail to the 3' end of the coding sequence. All of these
sequences may be inserted into eukaryotic expression vectors.

[0303] Other promoters suitable for use with prokaryotic hosts include the
phoA promoter, -lactamase and lactose promoter systems, alkaline
phosphatase promoter, a tryptophan (trp) promoter system, and hybrid
promoters such as the tac promoter. However, other known bacterial
promoters are suitable. Promoters for use in bacterial systems also will
contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA
encoding the antibody polypeptide.

[0304] Antibody polypeptide transcription from vectors in mammalian host
cells is controlled, for example, by promoters obtained from the genomes
of viruses such as polyoma virus, fowlpox virus, adenovirus (such as
Adenovirus 2), bovine papilloma virus, avian sarcoma virus,
cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably
Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the
actin promoter or an immunoglobulin promoter, from heat-shock promoters,
provided such promoters are compatible with the host cell systems.

[0305] The early and late promoters of the SV40 virus are conveniently
obtained as an SV40 restriction fragment that also contains the SV40
viral origin of replication. The immediate early promoter of the human
cytomegalovirus is conveniently obtained as a HindIII E restriction
fragment. A system for expressing DNA in mammalian hosts using the bovine
papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446. A
modification of this system is described in U.S. Pat. No. 4,601,978. See
also Reyes et al., Nature 297:598-601 (1982) on expression of
human-interferon cDNA in mouse cells under the control of a thymidine
kinase promoter from herpes simplex virus. Alternatively, the Rous
Sarcoma Virus long terminal repeat can be used as the promoter.

[0306] e) Enhancer Element Component

[0307] Transcription of a DNA encoding the antibodies of this invention by
higher eukaryotes is often increased by inserting an enhancer sequence
into the vector. Many enhancer sequences are now known from mammalian
genes (globin, elastase, albumin, α-fetoprotein, and insulin).
Typically, however, one will use an enhancer from a eukaryotic cell
virus. Examples include the SV40 enhancer on the late side of the
replication origin (bp 100-270), the cytomegalovirus early promoter
enhancer, the polyoma enhancer on the late side of the replication
origin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982)
on enhancing elements for activation of eukaryotic promoters. The
enhancer may be spliced into the vector at a position 5' or 3' to the
antibody encoding sequence, but is preferably located at a site 5' from
the promoter.

[0308] f) Transcription Termination Component

[0309] Expression vectors used in eukaryotic host cells (yeast, fungi,
insect, plant, animal, human, or nucleated cells from other multicellular
organisms) will also contain sequences necessary for the termination of
transcription and for stabilizing the mRNA. Such sequences are commonly
available from the 5' and, occasionally 3', untranslated regions of
eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide
segments transcribed as polyadenylated fragments in the untranslated
portion of the antibody-encoding mRNA. One useful transcription
termination component is the bovine growth hormone polyadenylation
region. See WO94/11026 and the expression vector disclosed therein.

[0312] Host cells are transformed with the above-described expression or
cloning vectors for antibody production and cultured in conventional
nutrient media modified as appropriate for inducing promoters, selecting
transformants, or amplifying the genes encoding the desired sequences.
Examples of useful mammalian host celllines are

[0313] h) Culturing the Host Cells

[0314] The host cells used to produce the antibody of this invention may
be cultured in a variety of media. Commercially available media such as
Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640
(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are
suitable for culturing the host cells. In addition, any of the media
described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.
Biochem. 102:255 (1980), U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762;
4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Patent Re.
30,985 may be used as culture media for the host cells. Any of these
media may be supplemented as necessary with hormones and/or other growth
factors (such as insulin, transferrin, or epidermal growth factor), salts
(such as sodium chloride, calcium, magnesium, and phosphate), buffers
(such as HEPES), nucleotides (such as adenosine and thymidine),
antibiotics (such as GENTAMYCIN® drug), trace elements (defined as
inorganic compounds usually present at final concentrations in the
micromolar range), and glucose or an equivalent energy source. Any other
necessary supplements may also be included at appropriate concentrations
that would be known to those skilled in the art. The culture conditions,
such as temperature, pH, and the like, are those previously used with the
host cell selected for expression, and will be apparent to the ordinarily
skilled artisan.

[0315] i) Purification of Antibody

[0316] When using recombinant techniques, the antibody can be produced
intracellularly, in the periplasmic space, or directly secreted into the
medium. If the antibody is produced intracellularly, as a first step, the
particulate debris, either host cells or lysed fragments, are removed,
for example, by centrifugation or ultrafiltration. Carter et al.,
Bio/Technology 10:163-167 (1992) describe a procedure for isolating
antibodies which are secreted to the periplasmic space of E. coli.
Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5),
EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell
debris can be removed by centrifugation. Where the antibody is secreted
into the medium, supernatants from such expression systems are generally
first concentrated using a commercially available protein concentration
filter, for example, an Amicon or Millipore Pellicon ultrafiltration
unit. A protease inhibitor such as PMSF may be included in any of the
foregoing steps to inhibit proteolysis and antibiotics may be included to
prevent the growth of adventitious contaminants.

[0317] The antibody composition prepared from the cells can be purified
using, for example, hydroxylapatite chromatography, gel electrophoresis,
dialysis, and affinity chromatography, with affinity chromatography being
the preferred purification technique. The suitability of protein A as an
affinity ligand depends on the species and isotype of any immunoglobulin
Fc domain that is present in the antibody. Protein A can be used to
purify antibodies that are based on human immunoglobulins containing 1,
2, or 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)).
Protein G is recommended for all mouse isotypes and for human 3 (Guss et
al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand
is attached is most often agarose, but other matrices are available.
Mechanically stable matrices such as controlled pore glass or
poly(styrene-divinyl)benzene allow for faster flow rates and shorter
processing times than can be achieved with agarose. Where the antibody
comprises a CH3 domain, the Bakerbond ABX®resin (J. T. Baker,
Phillipsburg, N.J.) is useful for purification. Other techniques for
protein purification such as fractionation on an ion-exchange column,
ethanol precipitation, Reverse Phase HPLC, chromatography on silica,
chromatography on heparin SEPHAROSE® chromatography on an anion or
cation exchange resin (such as a polyaspartic acid column),
chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also
available depending on the antibody to be recovered.

[0318] Following any preliminary purification step(s), the mixture
comprising the antibody of interest and contaminants may be subjected to
low pH hydrophobic interaction chromatography using an elution buffer at
a pH between about 2.5-4.5, preferably performed at low salt
concentrations (e.g., from about 0-0.25M salt).

[0319] C. Antibody Preparation

[0320] 1) Polyclonal Antibodies

[0321] Polyclonal antibodies are generally raised in animals by multiple
subcutaneous (sc) or intraperitoneal (ip) injections of the relevant
antigen and an adjuvant. It may be useful to conjugate the relevant
antigen to a protein that is immunogenic in the species to be immunized,
e.g., keyhole limpet hemocyanin (KLH), serum albumin, bovine
thyroglobulin, or soybean trypsin inhibitor, using a bifunctional or
derivatizing agent, e.g., maleimidobenzoyl sulfosuccinimide ester
(conjugation through cysteine residues), N-hydroxysuccinimide (through
lysien residues), glutaraldehyde, succinic anhydride, SOCl2, or
R1N═C═NR, where R and R1 are independently lower alkyl
groups. Examples of adjuvants which may be employed include Freund's
complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic
trehalose dicorynomycolate). The immunization protocol may be selected by
one skilled in the art without undue experimentation.

[0322] The animals are immunized against the antigen, immunogenic
conjugates, or derivatives by combining, e.g., 100 μg or 5 μg or
the protein or conjugate (for rabbits or mice, respectively) with 3
volumes of Freund's complete adjuvant and injecting the solution
intradermally at multiple sites. One month later, the animals are boosted
with 1/5 to 1/10 the original amount of peptide or conjugate in Freund's
complete adjuvant by subcutaneous injection at multiple sites. Seven to
fourteen days later, the animals are bled and the serum is assayed for
antibody titer. Animals are boosted until the titer plateaus. Conjugates
also can be made in recombinant cell culture as protein fusions. Also,
aggregating agents such as alum are suitable to enhance the immune
response.

[0323] 2) Monoclonal Antibodies

[0324] Monoclonal antibodies are obtained from a population of
substantially homogeneous antibodies, i.e., the individual antibodies
comprising the population are identical except for possible naturally
occurring mutations and/or post-translational modifications (e.g.,
isomerizations, amidations) that may be present in minor amounts. Thus,
the modifier "monoclonal" indicates the character of the antibody as not
being a mixture of discrete antibodies.

[0325] For example, the monoclonal antibodies may be made using the
hybridoma method first described by Kohler et al., Nature, 256:495
(1975), or may be made by recombinant DNA methods (U.S. Pat. No.
4,816,567).

[0326] In the hybridoma method, a mouse or other appropriate host animal,
such as a hamster, is immunized as hereinabove described to elicit
lymphocytes that produce or are capable of producing antibodies that will
specifically bind to the protein used for immunization. Alternatively,
lymphocytes may be immunized in vitro. Lymphocytes then are fused with
myeloma cells using a suitable fusing agent, such as polyethylene glycol,
to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and
Practice, pp. 59-103 (Academic Press, 1986).

[0327] The immunizing agent will typically include the antigenic protein
or a fusion variant thereof. Generally either peripheral blood
lymphocytes ("PBLs") are used if cells of human origin are desired, or
spleen cells or lymph node cells are used if non-human mammalian sources
are desired. The lymphocytes are then fused with an immortalized cell
line using a suitable fusing agent, such as polyethylene glycol, to form
a hybridoma cell. Goding, Monoclonal Antibodies: Principles and Practice,
Academic Press (1986), pp. 59-103.

[0328] Immortalized cell lines are usually transformed mammalian cells,
particularly myeloma cells of rodent, bovine and human origin. Usually,
rat or mouse myeloma cell lines are employed. The hybridoma cells thus
prepared are seeded and grown in a suitable culture medium that
preferably contains one or more substances that inhibit the growth or
survival of the unfused, parental myeloma cells. For example, if the
parental myeloma cells lack the enzyme hypoxanthine guanine
phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the
hybridomas typically will include hypoxanthine, aminopterin, and
thymidine (HAT medium), which are substances that prevent the growth of
HGPRT-deficient cells.

[0329] Preferred immortalized myeloma cells are those that fuse
efficiently, support stable high-level production of antibody by the
selected antibody-producing cells, and are sensitive to a medium such as
HAT medium. Among these, preferred are murine myeloma lines, such as
those derived from MOPC-21 and MPC-11 mouse tumors available from the
Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2
cells (and derivatives thereof, e.g., X63-Ag8-653) available from the
American Type Culture Collection, Manassas, Va. USA. Human myeloma and
mouse-human heteromyeloma cell lines also have been described for the
production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001
(1984); Brodeur et al., Monoclonal Antibody Production Techniques and
Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0330] Culture medium in which hybridoma cells are growing is assayed for
production of monoclonal antibodies directed against the antigen.
Preferably, the binding specificity of monoclonal antibodies produced by
hybridoma cells is determined by immunoprecipitation or by an in vitro
binding assay, such as radioimmunoassay (RIA) or enzyme-linked
immunosorbent assay (ELISA).

[0331] The culture medium in which the hybridoma cells are cultured can be
assayed for the presence of monoclonal antibodies directed against the
desired antigen. Preferably, the binding affinity and specificity of the
monoclonal antibody can be determined by immunoprecipitation or by an in
vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked
assay (ELISA). Such techniques and assays are known in the in art. For
example, binding affinity may be determined by the Scatchard analysis of
Munson et al., Anal. Biochem., 107:220 (1980).

[0332] After hybridoma cells are identified that produce antibodies of the
desired specificity, affinity, and/or activity, the clones may be
subcloned by limiting dilution procedures and grown by standard methods
(Goding, supra). Suitable culture media for this purpose include, for
example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may
be grown in vivo as tumors in a mammal.

[0334] Monoclonal antibodies may also be made by recombinant DNA methods,
such as those described in U.S. Pat. No. 4,816,567, and as described
above. DNA encoding the monoclonal antibodies is readily isolated and
sequenced using conventional procedures (e.g., by using oligonucleotide
probes that are capable of binding specifically to genes encoding the
heavy and light chains of murine antibodies). The hybridoma cells serve
as a preferred source of such DNA. Once isolated, the DNA may be placed
into expression vectors, which are then transfected into host cells such
as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein, in
order to synthesize monoclonal antibodies in such recombinant host cells.
Review articles on recombinant expression in bacteria of DNA encoding the
antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262
(1993) and Pluckthun, Immunol. Revs. 130:151-188 (1992).

[0335] In a further embodiment, antibodies can be isolated from antibody
phage libraries generated using the techniques described in McCafferty et
al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628
(1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the
isolation of murine and human antibodies, respectively, using phage
libraries. Subsequent publications describe the production of high
affinity (nM range) human antibodies by chain shuffling (Marks et al.,
Bio/Technology, 10:779-783 (1992)), as well as combinatorial infection
and in vivo recombination as a strategy for constructing very large phage
libraries (Waterhouse et al., Nucl. Acids Res., 21:2265-2266 (1993)).
Thus, these techniques are viable alternatives to traditional monoclonal
antibody hybridoma techniques for isolation of monoclonal antibodies.

[0336] The DNA also may be modified, for example, by substituting the
coding sequence for human heavy- and light-chain constant domains in
place of the homologous murine sequences (U.S. Pat. No. 4,816,567;
Morrison, et al., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by
covalently joining to the immunoglobulin coding sequence all or part of
the coding sequence for a non-immunoglobulin polypeptide. Typically such
non-immunoglobulin polypeptides are substituted for the constant domains
of an antibody, or they are substituted for the variable domains of one
antigen-combining site of an antibody to create a chimeric bivalent
antibody comprising one antigen-combining site having specificity for an
antigen and another antigen-combining site having specificity for a
different antigen.

[0337] The monoclonal antibodies described herein may by monovalent, the
preparation of which is well known in the art. For example, one method
involves recombinant expression of immunoglobulin light chain and a
modified heavy chain. The heavy chain is truncated generally at any point
in the Fc region so as to prevent heavy chain crosslinking.
Alternatively, the relevant cysteine residues may be substituted with
another amino acid residue or are deleted so as to prevent crosslinking.
In vitro methods are also suitable for preparing monovalent antibodies.
Digestion of antibodies to produce fragments thereof, particularly Fab
fragments, can be accomplished using routine techniques known in the art.

[0338] Chimeric or hybrid antibodies also may be prepared in vitro using
known methods in synthetic protein chemistry, including those involving
crosslinking agents. For example, immunotoxins may be constructed using a
disulfide-exchange reaction or by forming a thioether bond. Examples of
suitable reagents for this purpose include iminothiolate and
methyl-4-mercaptobutyrimidate.

[0339] 3) Humanized Antibodies.

[0340] The antibodies of the invention may further comprise humanized or
human antibodies. Humanized forms of non-human (e.g., murine) antibodies
are chimeric immunoglobulins, immunoglobulin chains or fragments thereof
(such as Fv, Fab, Fab', F(ab')2 or other antigen-binding
subsequences of antibodies) which contain minimal sequence derived from
non-human immunoglobulin. Humanized antibodies include human
immunoglobulins (recipient antibody) in which residues from a
complementarity determining region (CDR) (HVR as used herein) of the
recipient are replaced by residues from a CDR of a non-human species
(donor antibody) such as mouse, rat or rabbit having the desired
specificity, affinity and capacity. In some instances, Fv framework
residues of the human immunoglobulin are replaced by corresponding
non-human residues. Humanized antibodies may also comprise residues which
are found neither in the recipient antibody nor in the imported CDR or
framework sequences. In general, the humanized antibody will comprise
substantially all of at least one, and typically two, variable domain, in
which all or substantially all of the CDR regions correspond to those of
a non-human immunoglobulin and all or substantially all of the FR regions
are those of a human immunoglobulin consensus sequence. The humanized
antibody optimally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. Jones et al., Nature 321: 522-525 (1986); Riechmann et
al., Nature 332: 323-329 (1988) and Presta, Curr. Opin. Struct. Biol. 2:
593-596 (1992).

[0341] Methods for humanizing non-human antibodies are well known in the
art. Generally, a humanized antibody has one or more amino acid residues
introduced into it from a source which is non-human. These non-human
amino acid residues are often referred to as "import" residues, which are
typically taken from an "import" variable domain. Humanization can be
essentially performed following the method of Winter and co-workers,
Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature
332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988), or
through substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized" antibodies
are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially
less than an intact human variable domain has been substituted by the
corresponding sequence from a non-human species. In practice, humanized
antibodies are typically human antibodies in which some CDR residues and
possibly some FR residues are substituted by residues from analogous
sites in rodent antibodies.

[0342] The choice of human variable domains, both light and heavy, to be
used in making the humanized antibodies is very important to reduce
antigenicity. According to the so-called "best-fit" method, the sequence
of the variable domain of a rodent antibody is screened against the
entire library of known human variable-domain sequences. The human
sequence which is closest to that of the rodent is then accepted as the
human framework (FR) for the humanized antibody. Sims et al., J.
Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987).
Another method uses a particular framework derived from the consensus
sequence of all human antibodies of a particular subgroup of light or
heavy chains. The same framework may be used for several different
humanized antibodies. Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285
(1992); Presta et al., J. Immunol., 151:2623 (1993).

[0343] It is further important that antibodies be humanized with retention
of high affinity for the antigen and other favorable biological
properties. To achieve this goal, according to a preferred method,
humanized antibodies are prepared by a process of analysis of the
parental sequences and various conceptual humanized products using
three-dimensional models of the parental and humanized sequences.
Three-dimensional immunoglobulin models are commonly available and are
familiar to those skilled in the art. Computer programs are available
which illustrate and display probable three-dimensional conformational
structures of selected candidate immunoglobulin sequences. Inspection of
these displays permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the analysis
of residues that influence the ability of the candidate immunoglobulin to
bind its antigen. In this way, FR residues can be selected and combined
from the recipient and import sequences so that the desired antibody
characteristic, such as increased affinity for the target antigen(s), is
achieved. In general, the CDR residues are directly and most
substantially involved in influencing antigen binding.

[0344] Various forms of the humanized antibody are contemplated. For
example, the humanized antibody may be an antibody fragment, such as an
Fab, which is optionally conjugated with one or more cytotoxic agent(s)
in order to generate an immunoconjugate. Alternatively, the humanized
antibody may be an intact antibody, such as an intact IgG1 antibody.

[0345] 4) Human Antibodies

[0346] As an alternative to humanization, human antibodies can be
generated. For example, it is now possible to produce transgenic animals
(e.g., mice) that are capable, upon immunization, of producing a full
repertoire of human antibodies in the absence of endogenous
immunoglobulin production. For example, it has been described that the
homozygous deletion of the antibody heavy-chain joining region (JH)
gene in chimeric and germ-line mutant mice results in complete inhibition
of endogenous antibody production. Transfer of the human germ-line
immunoglobulin gene array in such germ-line mutant mice will result in
the production of human antibodies upon antigen challenge. See, e.g.,
Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits
et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immuno.,
7:33 (1993); U.S. Pat. No. 5,591,669 and WO 97/17852.

[0347] Alternatively, phage display technology can be used to produce
human antibodies and antibody fragments in vitro, from immunoglobulin
variable (V) domain gene repertoires from unimmunized donors. McCafferty
et al., Nature 348:552-553 (1990); Hoogenboom and Winter, J. Mol. Biol.
227: 381 (1991). According to this technique, antibody V domain genes are
cloned in-frame into either a major or minor coat protein gene of a
filamentous bacteriophage, such as M13 or fd, and displayed as functional
antibody fragments on the surface of the phage particle. Because the
filamentous particle contains a single-stranded DNA copy of the phage
genome, selections based on the functional properties of the antibody
also result in selection of the gene encoding the antibody exhibiting
those properties. Thus, the phage mimics some of the properties of the
B-cell. Phage display can be performed in a variety of formats, reviewed
in, e.g., Johnson, Kevin S. and Chiswell, David J., Curr. Opin Struct.
Biol. 3:564-571 (1993). Several sources of V-gene segments can be used
for phage display. Clackson et al., Nature 352:624-628 (1991) isolated a
diverse array of anti-oxazolone antibodies from a small random
combinatorial library of V genes derived from the spleens of immunized
mice. A repertoire of V genes from unimmunized human donors can be
constructed and antibodies to a diverse array of antigens (including
self-antigens) can be isolated essentially following the techniques
described by Marks et al., J. Mol. Biol. 222:581-597 (1991), or Griffith
et al., EMBO J. 12:725-734 (1993). See also, U.S. Pat. Nos. 5,565,332 and
5,573,905.

[0349] Finally, human antibodies may also be generated in vitro by
activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

[0350] 5) Antibody Fragments

[0351] In certain circumstances there are advantages to using antibody
fragments, rather than whole antibodies. Smaller fragment sizes allow for
rapid clearance, and may lead to improved access to solid tumors.

[0352] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., J
Biochem Biophys. Method. 24:107-117 (1992); and Brennan et al., Science
229:81 (1985)). However, these fragments can now be produced directly by
recombinant host cells. Fab, Fv and scFv antibody fragments can all be
expressed in and secreted from E. coli, thus allowing the facile
production of large amounts of these fragments. Antibody fragments can be
isolated from the antibody phage libraries discussed above.
Alternatively, Fab'-SH fragments can be directly recovered from E. coli
and chemically coupled to form F(ab')2 fragments (Carter et al.,
Bio/Technology 10:163-167 (1992)). According to another approach,
F(ab')2 fragments can be isolated directly from recombinant host
cell culture. Fab and F(ab')2 with increase in vivo half-life is
described in U.S. Pat. No. 5,869,046. In other embodiments, the antibody
of choice is a single chain Fv fragment (scFv). See WO 93/16185; U.S.
Pat. No. 5,571,894 and U.S. Pat. No. 5,587,458. The antibody fragment may
also be a "linear antibody", e.g., as described in U.S. Pat. No.
5,641,870. Such linear antibody fragments may be monospecific or
bispecific.

[0353] 6) Antibody Dependent Enzyme-Mediated Prodrug Therapy (ADEPT)

[0354] The antibodies of the present invention may also be used in ADEPT
by conjugating the antibody to a prodrug-activating enzyme which converts
a prodrug (e.g. a peptidyl chemotherapeutic agent, see WO 81/01145) to an
active anti-cancer drug. See, for example, WO 88/07378 and U.S. Pat. No.
4,975,278.

[0355] The enzyme component of the immunoconjugate useful for ADEPT
includes any enzyme capable of acting on a prodrug in such a way so as to
convert it into its more active, cytotoxic form.

[0356] Enzymes that are useful in the method of this invention include,
but are not limited to, glycosidase, glucose oxidase, human lysozyme,
human glucuronidase, alkaline phosphatase useful for converting
phosphate-containing prodrugs into free drugs; arylsulfatase useful for
converting sulfate-containing prodrugs into free drugs; cytosine
deaminase useful for converting non-toxic 5-fluorocytosine into the
anti-cancer drug 5-fluorouracil; proteases, such as serratia protease,
thermolysin, subtilisin, carboxypeptidases (e.g., carboxypeptidase G2 and
carboxypeptidase A) and cathepsins (such as cathepsins B and L), that are
useful for converting peptide-containing prodrugs into free drugs;
D-alanylcarboxypeptidases, useful for converting prodrugs that contain
D-amino acid substituents; carbohydrate-cleaving enzymes such as
β-galactosidase and neuraminidase useful for converting glycosylated
prodrugs into free drugs; β-lactamase useful for converting drugs
derivatized with β-lactams into free drugs; and penicillin amidases,
such as penicillin Vamidase or penicillin G amidase, useful for
converting drugs derivatized at their amine nitrogens with phenoxyacetyl
or phenylacetyl groups, respectively, into free drugs. Alternatively,
antibodies with enzymatic activity, also known in the art as "abzymes"
can be used to convert the prodrugs of the invention into free active
drugs (see, e.g., Massey, Nature 328: 457-458 (1987)). Antibody-abzyme
conjugates can be prepared as described herein for delivery of the abzyme
to a tumor cell population.

[0357] The above enzymes can be covalently bound to the polypeptide or
antibodies described herein by techniques well known in the art such as
the use of the heterobifunctional cross-linking agents discussed above.
Alternatively, fusion proteins comprising at least the antigen binding
region of the antibody of the invention linked to at least a functionally
active portion of an enzyme of the invention can be constructed using
recombinant DNA techniques well known in the art (see, e.g. Neuberger et
al., Nature 312: 604-608 (1984)).

[0358] 7) Bispecific and Polyspecific Antibodies

[0359] Bispecific antibodies (BsAbs) are antibodies that have binding
specificities for at least two different epitopes, including those on the
same or another protein. Alternatively, one arm can bind to the target
antigen, and another arm can be combined with an arm that binds to a
triggering molecule on a leukocyte such as a T-cell receptor molecule
(e.g., CD3), or Fc receptors for IgG (FcγR) such as FcγR1
(CD64), FcγRII (CD32) and FcγRIII (CD16), so as to focus and
localize cellular defense mechanisms to the target antigen-expressing
cell. Such antibodies can be derived from full length antibodies or
antibody fragments (e.g. F(ab')2 bispecific antibodies).

[0360] Bispecific antibodies may also be used to localize cytotoxic agents
to cells which express the target antigen. Such antibodies possess one
arm that binds the desired antigen and another arm that binds the
cytotoxic agent (e.g., saporin, anti-interferon-α, vinca alkoloid,
ricin A chain, methotrexate or radioactive isotope hapten). Examples of
known bispecific antibodies include anti-ErbB2/anti-FcgRIII (WO
96/16673), anti-ErbB2/anti-FcgRI (U.S. Pat. No. 5,837,234),
anti-ErbB2/anti-CD3 (U.S. Pat. No. 5,821,337).

[0361] Methods for making bispecific antibodies are known in the art.
Traditional production of full length bispecific antibodies is based on
the coexpression of two immunoglobulin heavy-chain/light chain pairs,
where the two chains have different specificities. Millstein et al.,
Nature, 305:537-539 (1983). Because of the random assortment of
immunoglobulin heavy and light chains, these hybridomas (quadromas)
produce a potential mixture of 10 different antibody molecules, of which
only one has the correct bispecific structure. Purification of the
correct molecule, which is usually done by affinity chromatography steps,
is rather cumbersome, and the product yields are low. Similar procedures
are disclosed in WO 93/08829 and in Traunecker et al., EMBO J.,
10:3655-3659 (1991).

[0362] According to a different approach, antibody variable domains with
the desired binding specificities (antibody-antigen combining sites) are
fused to immunoglobulin constant domain sequences. The fusion preferably
is with an immunoglobulin heavy chain constant domain, comprising at
least part of the hinge, CH2, and CH3 regions. It is preferred to have
the first heavy-chain constant region (CH1) containing the site necessary
for light chain binding, present in at least one of the fusions. DNAs
encoding the immunoglobulin heavy chain fusions and, if desired, the
immunoglobulin light chain, are inserted into separate expression
vectors, and are co-transfected into a suitable host organism. This
provides for great flexibility in adjusting the mutual proportions of the
three polypeptide fragments in embodiments when unequal ratios of the
three polypeptide chains used in the construction provide the optimum
yields. It is, however, possible to insert the coding sequences for two
or all three polypeptide chains in one expression vector when the
expression of at least two polypeptide chains in equal ratios results in
high yields or when the ratios are of no particular significance.

[0363] In a preferred embodiment of this approach, the bispecific
antibodies are composed of a hybrid immunoglobulin heavy chain with a
first binding specificity in one arm, and a hybrid immunoglobulin heavy
chain-light chain pair (providing a second binding specificity) in the
other arm. It was found that this asymmetric structure facilitates the
separation of the desired bispecific compound from unwanted
immunoglobulin chain combinations, as the presence of an immunoglobulin
light chain in only one half of the bispecific molecules provides for an
easy way of separation. This approach is disclosed in WO 94/04690. For
further details of generating bispecific antibodies, see, for example,
Suresh et al., Methods in Enzymology 121: 210 (1986).

[0364] According to another approach described in WO 96/27011 or U.S. Pat.
No. 5,731,168, the interface between a pair of antibody molecules can be
engineered to maximize the percentage of heterodimers which are recovered
from recombinant cell culture. The preferred interface comprises at least
a part of the CH3 region of an antibody constant domain. In this method,
one or more small amino acid side chains from the interface of the first
antibody molecule are replaced with larger side chains (e.g., tyrosine or
tryptophan). Compensatory "cavities" of identical or similar size to the
large side chains(s) are created on the interface of the second antibody
molecule by replacing large amino acid side chains with smaller ones
(e.g., alanine or threonine). This provides a mechanism for increasing
the yield of the heterodimer over other unwanted end-products such as
homodimers.

[0365] Techniques for generating bispecific antibodies from antibody
fragments have been described in the literature. For example, bispecific
antibodies can be prepared using chemical linkage. Brennan et al.,
Science 229: 81 (1985) describe a procedure wherein intact antibodies are
proteolytically cleaved to generate F(ab')2 fragments. These
fragments are reduced in the presence of the dithiol complexing agent
sodium arsenite to stabilize vicinal dithiols and prevent intermolecular
disulfide formation. The Fab' fragments generated are then converted to
thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is
then reconverted to the Fab'-TNB derivative to form the bispecific
antibody. The bispecific antibodies produced can be used as agents for
the selective immobilization of enzymes.

[0366] Fab' fragments may be directly recovered from E. coli and
chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp.
Med. 175: 217-225 (1992) describes the production of fully humanized
bispecific antibody F(ab')2 molecules. Each Fab' fragment was
separately secreted from E. coli and subjected to directed chemical
coupling in vitro to form the bispecific antibody. The bispecific
antibody thus formed was able to bind to cells overexpressing the ErbB2
receptor and normal human T cells, as well as trigger the lytic activity
of human cytotoxic lymphocytes against human breast tumor targets.

[0367] Various techniques for making and isolating bivalent antibody
fragments directly from recombinant cell culture have also been
described. For example, bivalent heterodimers have been produced using
leucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).
The leucine zipper peptides from the Fos and Jun proteins were linked to
the Fab' portions of two different antibodies by gene fusion. The
antibody homodimers were reduced at the hinge region to form monomers and
then re-oxidized to form the antibody heterodimers. The "diabody"
technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:
6444-6448 (1993) has provided an alternative mechanism for making
bispecific/bivalent antibody fragments. The fragments comprise a
heavy-chain variable domain (VH) connected to a light-chain variable
domain (VL) by a linker which is too short to allow pairing between
the two domains on the same chain. Accordingly, the VH and VL
domains of one fragment are forced to pair with the complementary VL
and VH domains of another fragment, thereby forming two
antigen-binding sites. Another strategy for making bispecific/bivalent
antibody fragments by the use of single-chain Fv (sFv) dimers has also
been reported. See Gruber et al., J. Immunol., 152:5368 (1994).

[0368] Antibodies with more than two valencies are contemplated. For
example, trispecific antibodies can be prepared. Tutt et al., J. Immunol.
147: 60 (1991).

[0369] Exemplary bispecific antibodies may bind to two different epitopes
on a given molecule. Alternatively, an anti-protein arm may be combined
with an arm which binds to a triggering molecule on a leukocyte such as a
T-cell receptor molecule (e.g., CD2, CD3, CD28 or B7), or Fc receptors
for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and
FcγRIII (CD16) so as to focus cellular defense mechanisms to the
cell expressing the particular protein. Bispecific antibodies may also be
used to localize cytotoxic agents to cells which express a particular
protein. Such antibodies possess a protein-binding arm and an arm which
binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA,
DOTA or TETA. Another bispecific antibody of interest binds the protein
of interest and further binds tissue factor (TF).

[0370] 8) Multivalent Antibodies

[0371] A multivalent antibody may be internalized (and/or catabolized)
faster than a bivalent antibody by a cell expressing an antigen to which
the antibodies bind. The antibodies of the present invention can be
multivalent antibodies (which are other than of the IgM class) with three
or more antigen binding sites (e.g. tetravalent antibodies), which can be
readily produced by recombinant expression of nucleic acid encoding the
polypeptide chains of the antibody. The multivalent antibody can comprise
a dimerization domain and three or more antigen binding sites. The
preferred dimerization domain comprises (or consists of) an Fc region or
a hinge region. In this scenario, the antibody will comprise an Fc region
and three or more antigen binding sites amino-terminal to the Fc region.
The preferred multivalent antibody herein comprises (or consists of)
three to about eight, but preferably four, antigen binding sites. The
multivalent antibody comprises at least one polypeptide chain (and
preferably two polypeptide chains), wherein the polypeptide chain(s)
comprise two or more variable domains. For instance, the polypeptide
chain(s) may comprise VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a
first variable domain, VD2 is a second variable domain, Fc is one
polypeptide chain of an Fc region, X1 and X2 represent an amino acid or
polypeptide, and n is 0 or 1. For instance, the polypeptide chain(s) may
comprise: VH-CH1-flexible linker-VH-CH1-Fc region chain; or
VH-CH1-VH-CH1-Fc region chain. The multivalent antibody herein preferably
further comprises at least two (and preferably four) light chain variable
domain polypeptides. The multivalent antibody herein may, for instance,
comprise from about two to about eight light chain variable domain
polypeptides. The light chain variable domain polypeptides contemplated
here comprise a light chain variable domain and, optionally, further
comprise a CL domain.

[0372] 9) Heteroconjugate Antibodies

[0373] Heteroconjugate antibodies are also within the scope of the present
invention. Heteroconjugate antibodies are composed of two covalently
joined antibodies. For example, one of the antibodies in the
heteroconjugate can be coupled to avidin, the other to biotin. Such
antibodies have, for example, been proposed to target immune system cells
to unwanted cells, U.S. Pat. No. 4,676,980, and for treatment of HIV
infection. WO 91/00360, WO 92/200373 and EP 0308936. It is contemplated
that the antibodies may be prepared in vitro using known methods in
synthetic protein chemistry, including those involving crosslinking
agents. For example, immunotoxins may be constructed using a disulfide
exchange reaction or by forming a thioether bond. Examples of suitable
reagents for this purpose include iminothiolate and
methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S.
Pat. No. 4,676,980. Heteroconjugate antibodies may be made using any
convenient cross-linking methods. Suitable cross-linking agents are well
known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along
with a number of cross-linking techniques.

[0374] 10) Effector Function Engineering

[0375] It may be desirable to modify the antibody of the invention with
respect to Fc effector function, e.g., so as to modify (e.g., enhance or
eliminate) antigen-dependent cell-mediated cyotoxicity (ADCC) and/or
complement dependent cytotoxicity (CDC) of the antibody. In a preferred
embodiment, Fc effector function of the anti-PD-L1 antibodies is reduced
or eliminated. This may be achieved by introducing one or more amino acid
substitutions in an Fc region of the antibody. Alternatively or
additionally, cysteine residue(s) may be introduced in the Fc region,
thereby allowing interchain disulfide bond formation in this region. The
homodimeric antibody thus generated may have improved internalization
capability and/or increased complement-mediated cell killing and
antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp
Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922
(1992). Homodimeric antibodies with enhanced anti-tumor activity may also
be prepared using heterobifunctional cross-linkers as described in Wolff
et al., Cancer Research 53:2560-2565 (1993). Alternatively, an antibody
can be engineered which has dual Fc regions and may thereby have enhanced
complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer
Drug Design 3:219-230 (1989).

[0376] To increase the serum half life of the antibody, one may
incorporate a salvage receptor binding epitope into the antibody
(especially an antibody fragment) as described in U.S. Pat. No.
5,739,277, for example. As used herein, the term "salvage receptor
binding epitope" refers to an epitope of the Fc region of an IgG molecule
(e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible
for increasing the in vivo serum half-life of the IgG molecule.

[0377] 11) Other Amino Acid Sequence Modifications

[0378] Amino acid sequence modification(s) of the antibodies described
herein are contemplated. For example, it may be desirable to improve the
binding affinity and/or other biological properties of the antibody Amino
acid sequence variants of the antibody are prepared by introducing
appropriate nucleotide changes into the antibody nucleic acid, or by
peptide synthesis. Such modifications include, for example, deletions
from, and/or insertions into and/or substitutions of, residues within the
amino acid sequences of the antibody. Any combination of deletion,
insertion, and substitution is made to arrive at the final construct,
provided that the final construct possesses the desired characteristics.
The amino acid changes also may alter post-translational processes of the
antibody, such as changing the number or position of glycosylation sites.

[0379] A useful method for identification of certain residues or regions
of the antibody that are preferred locations for mutagenesis is called
"alanine scanning mutagenesis" as described by Cunningham and Wells in
Science, 244:1081-1085 (1989). Here, a residue or group of target
residues are identified (e.g., charged residues such as arg, asp, his,
lys, and glu) and replaced by a neutral or negatively charged amino acid
(most preferably alanine or polyalanine) to affect the interaction of the
amino acids antigen. Those amino acid locations demonstrating functional
sensitivity to the substitutions then are refined by introducing further
or other variants at, or for, the sites of substitution. Thus, while the
site for introducing an amino acid sequence variation is predetermined,
the nature of the mutation per se need not be predetermined. For example,
to analyze the performance of a mutation at a given site, ala scanning or
random mutagenesis is conducted at the target codon or region and the
expressed antibody variants are screened for the desired activity.

[0380] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an N-terminal
methionyl residue or the antibody fused to a cytotoxic polypeptide. Other
insertional variants of the antibody molecule include the fusion to the
N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a
polypeptide which increases the serum half-life of the antibody.

[0381] Another type of variant is an amino acid substitution variant.
These variants have at least one amino acid residue in the antibody
molecule replaced by a different residue. The sites of greatest interest
for substitutional mutagenesis include the hypervariable regions, but FR
alterations are also contemplated. Conservative substitutions are shown
in the Table A below under the heading of "preferred substitutions". If
such substitutions result in a change in biological activity, then more
substantial changes, denominated "exemplary substitutions" in Table A, or
as further described below in reference to amino acid classes, may be
introduced and the products screened.

[0382] Substantial modifications in the biological properties of the
antibody are accomplished by selecting substitutions that differ
significantly in their effect on maintaining (a) the structure of the
polypeptide backbone in the area of the substitution, for example, as a
sheet or helical conformation, (b) the charge or hydrophobicity of the
molecule at the target site, or (c) the bulk of the side chain. Naturally
occurring residues are divided into groups based on common side-chain
properties:

[0383] (1) hydrophobic: norleucine, met, ala, val, leu, ile;

[0384] (2) neutral hydrophilic: cys, ser, thr;

[0385] (3) acidic: asp, glu;

[0386] (4) basic: asn, gln, his, lys, arg;

[0387] (5) residues that influence chain orientation: gly, pro; and

[0388] (6) aromatic: trp, tyr, phe.

[0389] Non-conservative substitutions will entail exchanging a member of
one of these classes for another class.

[0390] Any cysteine residue not involved in maintaining the proper
conformation of the antibody also may be substituted, generally with
serine, to improve the oxidative stability of the molecule and prevent
aberrant crosslinking. Conversely, cysteine bond(s) may be added to the
antibody to improve its stability (particularly where the antibody is an
antibody fragment such as an Fv fragment).

[0391] A particularly preferred type of substitutional variant involves
substituting one or more hypervariable region residues of a parent
antibody (e.g. a humanized or human antibody). Generally, the resulting
variant(s) selected for further development will have improved biological
properties relative to the parent antibody from which they are generated.
A convenient way for generating such substitutional variants involves
affinity maturation using phage display. Briefly, several hypervariable
region sites (e.g. 6-7 sites) are mutated to generate all possible amino
substitutions at each site. The antibody variants thus generated are
displayed in a monovalent fashion from filamentous phage particles as
fusions to the gene III product of M13 packaged within each particle. The
phage-displayed variants are then screened for their biological activity
(e.g. binding affinity) as herein disclosed. In order to identify
candidate hypervariable region sites for modification, alanine scanning
mutagenesis can be performed to identify hypervariable region residues
contributing significantly to antigen binding. Alternatively, or
additionally, it may be beneficial to analyze a crystal structure of the
antigen-antibody complex to identify contact points between the antibody
and its target (e.g., PD-L1, B7.1). Such contact residues and neighboring
residues are candidates for substitution according to the techniques
elaborated herein. Once such variants are generated, the panel of
variants is subjected to screening as described herein and antibodies
with superior properties in one or more relevant assays may be selected
for further development.

[0392] Another type of amino acid variant of the antibody alters the
original glycosylation pattern of the antibody. By altering is meant
deleting one or more carbohydrate moieties found in the antibody, and/or
adding one or more glycosylation sites that are not present in the
antibody.

[0393] Glycosylation of antibodies is typically either N-linked or
O-linked. N-linked refers to the attachment of the carbohydrate moiety to
the side chain of an asparagine residue. The tripeptide sequences
asparagine-X-serine and asparagine-X-threonine, where X is any amino acid
except proline, are the recognition sequences for enzymatic attachment of
the carbohydrate moiety to the asparagine side chain. Thus, the presence
of either of these tripeptide sequences in a polypeptide creates a
potential glycosylation site. O-linked glycosylation refers to the
attachment of one of the sugars N-aceylgalactosamine, galactose, or
xylose to a hydroxyamino acid, most commonly serine or threonine,
although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0394] Addition of glycosylation sites to the antibody is conveniently
accomplished by altering the amino acid sequence such that it contains
one or more of the above-described tripeptide sequences (for N-linked
glycosylation sites). The alteration may also be made by the addition of,
or substitution by, one or more serine or threonine residues to the
sequence of the original antibody (for O-linked glycosylation sites).

[0395] Nucleic acid molecules encoding amino acid sequence variants to the
antibodies of the invention are prepared by a variety of methods known in
the art. These methods include, but are not limited to, isolation from a
natural source (in the case of naturally occurring amino acid sequence
variants) or preparation by oligonucleotide-mediated (or site-directed)
mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier
prepared variant or a non-variant versions.

[0396] 12) Other Antibody Modifications

[0397] The antibodies of the present invention can be further modified to
contain additional nonproteinaceous moieties that are known in the art
and readily available. Preferably, the moieties suitable for
derivatization of the antibody are water-soluble polymers. Non-limiting
examples of water-soluble polymers include, but are not limited to,
polyethylene glycol (PEG), copolymers of ethylene glycol/propylene
glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl
pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic
anhydride copolymer, polyaminoacids (either homopolymers or random
copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol,
polypropylene glycol homopolymers, polypropylene oxide/ethylene oxide
co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl
alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may
have advantages in manufacturing due to its stability in water. The
polymer may be of any molecular weight, and may be branched or
unbranched. The number of polymers attached to the antibody may vary, and
if more than one polymer is attached, they can be the same or different
molecules. In general, the number and/or type of polymers used for
derivatization can be determined based on considerations including, but
not limited to, the particular properties or functions of the antibody to
be improved, whether the antibody derivative will be used in a therapy
under defined conditions, etc. Such techniques and other suitable
formulations are disclosed in Remington: The Science and Practice of
Pharmacy, 20th Ed., Alfonso Gennaro, Ed., Philadelphia College of
Pharmacy and Science (2000).

[0400] When the therapeutic agent is an antibody fragment, the smallest
inhibitory fragment which specifically binds to the binding domain of the
target protein is preferred. For example, based upon the variable region
sequences of an antibody, antibody fragments or even peptide molecules
can be designed which retain the ability to bind the target protein
sequence. Such peptides can be synthesized chemically and/or produced by
recombinant DNA technology (see, e.g., Marasco et al., Proc. Natl. Acad.
Sci. USA 90: 7889-7893

[1993]).

[0401] Buffers are used to control the pH in a range which optimizes the
therapeutic effectiveness, especially if stability is pH dependent.
Buffers are preferably present at concentrations ranging from about 50 mM
to about 250 mM. Suitable buffering agents for use with the present
invention include both organic and inorganic acids and salts thereof. For
example, citrate, phosphate, succinate, tartrate, fumarate, gluconate,
oxalate, lactate, acetate. Additionally, buffers may be comprised of
histidine and trimethylamine salts such as Tris.

[0403] Tonicity agents, sometimes known as "stabilizers" are present to
adjust or maintain the tonicity of liquid in a composition. When used
with large, charged biomolecules such as proteins and antibodies, they
are often termed "stabilizers" because they can interact with the charged
groups of the amino acid side chains, thereby lessening the potential for
inter and intra-molecular interactions. Tonicity agents can be present in
any amount between 0.1% to 25% by weight, preferably 1 to 5%, taking into
account the relative amounts of the other ingredients. Preferred tonicity
agents include polyhydric sugar alcohols, preferably trihydric or higher
sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol
and mannitol.

[0405] Non-ionic surfactants or detergents (also known as "wetting
agents") are present to help solubilize the therapeutic agent as well as
to protect the therapeutic protein against agitation-induced aggregation,
which also permits the formulation to be exposed to shear surface stress
without causing denaturation of the active therapeutic protein or
antibody. Non-ionic surfactants are present in a range of about 0.05
mg/ml to about 1.0 mg/ml, preferably about 0.07 mg/ml to about 0.2 mg/ml.

[0407] The formulation may be rendered sterile by filtration through
sterile filtration membranes. The therapeutic compositions herein
generally are placed into a container having a sterile access port, for
example, an intravenous solution bag or vial having a stopper pierceable
by a hypodermic injection needle.

[0408] The route of administration is in accordance with known and
accepted methods, such as by single or multiple bolus or infusion over a
long period of time in a suitable manner, e.g., injection or infusion by
subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial,
intralesional or intraarticular routes, topical administration,
inhalation or by sustained release or extended-release means.

[0409] The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated,
preferably those with complementary activities that do not adversely
affect each other. Alternatively, or in addition, the composition may
comprise a cytotoxic agent, cytokine or growth inhibitory agent. Such
molecules are suitably present in combination in amounts that are
effective for the purpose intended.

[0410] The active ingredients may also be entrapped in microcapsules
prepared, for example, by coascervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsules and poly-(methylmethacylate) microcapsules,
respectively, in colloidal drug delivery systems (for example, liposomes,
albumin microspheres, microemulsions, nano-particles and nanocapsules) or
in macroemulsions. Such techniques are disclosed in Remington's
Pharmaceutical Sciences 18th edition, supra.

[0411] Stability of the proteins and antibodies described herein may be
enhanced through the use of non-toxic "water-soluble polyvalent metal
salts". Examples include Ca2+, Mg2+, Zn2+, Fe2+,
Fe3+, Cu2+, Sn2+, Sn4+, Al2+and Al3+.
Example anions that can form water soluble salts with the above
polyvalent metal cations include those formed from inorganic acids and/or
organic acids. Such water-soluble salts have a solubility in water (at
20° C.) of at least about 20 mg/ml, alternatively at least about
100 mg/ml, alternatively at least about 200 mg/ml.

[0412] Suitable inorganic acids that can be used to form the "water
soluble polyvalent metal salts" include hydrochloric, acetic, sulfuric,
nitric, thiocyanic and phosphoric acid. Suitable organic acids that can
be used include aliphatic carboxylic acid and aromatic acids. Aliphatic
acids within this definition may be defined as saturated or unsaturated
C2-9 carboxylic acids (e.g., aliphatic mono-, di- and tri-carboxylic
acids). For example, exemplary monocarboxylic acids within this
definition include the saturated C2-9 monocarboxylic acids acetic,
proprionic, butyric, valeric, caproic, enanthic, caprylic pelargonic and
capryonic, and the unsaturated C2-9 monocarboxylic acids acrylic,
propriolic methacrylic, crotonic and isocrotonic acids. Exemplary
dicarboxylic acids include the saturated C2-9 dicarboxylic acids
malonic, succinic, glutaric, adipic and pimelic, while unsaturated
C2-9 dicarboxylic acids include maleic, fumaric, citraconic and
mesaconic acids. Exemplary tricarboxylic acids include the saturated
C2-9 tricarboxylic acids tricarballylic and
1,2,3-butanetricarboxylic acid. Additionally, the carboxylic acids of
this definition may also contain one or two hydroxyl groups to form
hydroxy carboxylic acids. Exemplary hydroxy carboxylic acids include
glycolic, lactic, glyceric, tartronic, malic, tartaric and citric acid.
Aromatic acids within this definition include benzoic and salicylic acid.

[0415] For the prevention or treatment of disease, the appropriate dosage
of an active agent, will depend on the type of disease to be treated, as
defined above, the severity and course of the disease, whether the agent
is administered for preventive or therapeutic purposes, previous therapy,
the patient's clinical history and response to the agent, and the
discretion of the attending physician. The agent is suitably administered
to the patient at one time or over a series of treatments.

[0416] In a particular embodiment, the invention relates to costimulation
resulting from attenuating signaling through PD-1, specifically by the
application of PD-L1 antibodies that prevent binding to PD-1 and/or B7.1,
as well to the therapeutic treatment of T-cell dysfunctional disorders.

[0419] Microorganisms that cause chronic infection have exploited the
PD-1:PD-L signaling pathway to evade the host immune responses that
results in chronic infections. Viruses that cause chronic infection can
render virus-specific T cells non-functional and thereby silence the
antiviral T cell response. Barber et al., Nature 439: 682-87 (2006);
Wherry et al., J. Virol. 78: 5535-45 (2004). Exhaustion of T cells or
anergy, of CD8.sup.+ T cells is an important reason for ineffective viral
control during chronic infections and is characteristic of chronic LCMV
infections in mice as well as HIV, HBV, HCV and HTLV infection in human
and SW infection in primates. There appears to be a hierarchical,
progressive loss of function within the phenotype of exhausted
virus-specific CD8.sup.+ T cells, with cytotoxicity and IL-2 production
lost first, followed by effector cytokine production.

[0420] PD-1 is upregulated upon activation, and expression is maintained
at a high level by exhausted CD8.sup.+ T cells in mice with LCMV chronic
infection. Barber et al., supra. Administration of antibodies that
blocked PD-1: PD-L1 binding resulted in enhanced T cell responses and a
substantial reduction in viral burden. In persistently infected mice with
ineffective CD4.sup.+ TH response, blockade of PD-1:PD-L1 restored
CD8.sup.+ T cells from an dysfunctional state resulting in proliferation,
secretion of cytokines, killing of infected cells, and decreased viral
load, strongly suggesting a therapeutic approach for the treatment of
chronic viral infections.

[0423] The PD-1:PD-L pathway has also been implicated in the chronicity of
bacterial infections. Helicobacter pylori causes chronic gastritis and
gastroduodenal ulcers and is a risk factor for the development of gastric
cancer. During a H. pylori infection, T cell responses are insufficient
to clear infection, leading to persistent infection. Following exposure
to H. pylori in vitro or in vivo, PD-L1 is upregulated on gastric
epithelial cells. Gastric epithelial cells express MHC class II molecules
and are thought to play in important APC function during H. pylori
infection. Anti-PD-L1 antibodies that block PD-1 to PD-L1 interaction
enhance T cell proliferation and IL-2 production in cultures of gastric
epithelial cells explosed to H. pylori and CD4 T cells. Blocking PD-L1
with either antibodies or siRNA prevented the generation of the
regulatory T cells, suggesting that PD-L1 may promote T cell suppression
and persisting infections by controlling the dynamic between regulatory
and effector T cells during H. pylori infection. Beswick et al., Infect.
Immun. 75: 4334-41 (2007).

[0426] Empirical evidence for tumor immunity includes (i) the observance
of spontaneous remission, (ii) the presence of detectable, but
ineffective host immune response to tumors, (iii) the increased
prevalence of primary and secondary malignancies in immunodeficient
patients, (iv) the detection of increased levels of antibodies and
T-lymphocytes in tumor patients, and (v) the observation that test
animals can be immunized against various types of tumors.

[0434] Thus, the suppression of signaling through PD-L1 with the
anti-PD-L1 antibodies of the invention, so as to enhance T cell function,
shows promise to attenuate tumor immunity, and as a result, can be
effective treatment for cancer.

[0435] F. Combination Therapies

[0436] The method of the invention can be combined with known methods of
treatment chronic infection or cancer, either as combined or additional
treatment steps or as additional components of a therapeutic formulation.

[0437] 1. Cancer:

[0438] Enhancing the host's immune function to combat tumors is the
subject of increasing interest. Conventional methods include (i) APC
enchancement, such as (a) injection into the tumor of DNA encoding
foreign MHC alloantigens, or (b) transfecting biopsied tumor cells with
genes that increase the probability of immune antigen recognition (e.g.,
immune stimulatory cytokines, GM-CSF, co-stimulatory molecules B7.1,
B7.2) of the tumor, (iii) adoptive cellular immunotherapy, or treatment
with activated tumor-specific T-cells. Adoptive cellular immunotherapy
includes isolating tumor-infiltrating host T-lymphocytes, expanding the
population in vitro, such as through stimulation by IL-2 or tumor or
both. Additionally, isolated T-cells that are dysfunctional may be also
be activated by in vitro application of the anti-PD-L1 antibodies of the
invention. T-cells that are so-activated may then be readministered to
the host.

[0440] In the treatment of cancer, any of the previously described
conventional treatments for the treatment of cancer immunity may be
conducted, prior, subsequent or simultaneous with the administration of
the anti-PD-L1 antibodies of the invention. Additionally, the anti-PD-L1
antibodies of the invention may be administered prior, subsequent or
simultaneous with conventional cancer treatments, such as the
administration of tumor-binding antibodies (e.g., monoclonal antibodies,
toxin-conjugated monoclonal antibodies) and/or the administration of
chemotherapeutic agents.

[0441] 2. Infection:

[0442] In the treatment of infection (e.g., acute and/or chronic),
administration of the anti-PD-L1 antibodies of the invention can be
combined with conventional treatments in addition to or in lieu of
stimulating natural host immune defenses to infection. Natural host
immune defenses to infection include, but are not limited to
inflammation, fever, antibody-mediated host defense,
T-lymphocyte-mediated host defenses, including lymphokine secretion and
cytotoxic T-cells (especially during viral infection), complement
mediated lysis and opsonization (facilitated phagocytosis), and
phagocytosis. The ability of the anti-PD-L1 antibodies of the invention
to reactivate dysfunctional T-cells would be particularly useful to treat
chronic infections, in particular those in which cell-mediated immunity
is critical for complete recovery.

[0443] a. Bacteria

[0444] For infections resulting from a bacterial infection, the anti-PD-L1
antibodies of the invention may be combined by administration
simultaneous with, prior or subsequent to standard therapies for treating
bacterial infection. Bacterial infections are most commonly treated today
with antibacterial antibiotics, but serum containing pathogen-specific
antibodies from immunized hosts can also be effective.

[0445] Bacteria that are pathogenic as a result of the secretion of
toxins, (toxogenic bacteria), vaccination with inactive toxin and/or the
administration of therapeutic agents that block the toxicity of the
toxins are usually effective (e.g., polyclonal serum, antibodies,
antibiotics etc.). These organisms include Clostridium spp., bacillus
spp., Corynebacterium spp., Vibrio chloerae, Bordetella pertussis,
Staphylococcus spp., Streptococcus spp. Gram negative bacteria that also
typically respond to such traditional therapies include Enterobacteria
(E.g., Escherichia, Klebsiella, Proteus, Yersinia, Erwina), Salmonella,
and Pseudomonas aeruginosa. Encapsulated bacteria, which are resistant to
phagocytosis and opsonization, and thus often prevent a more significant
challenge to immune clearance include: Streptococcus spp., Haemophilus
spp. Neisseria spp., Klebsiella spp. and Bacterioides fragillis.

[0447] Spirochetes, including Treponema spp., Borrelia spp. and Leptospira
spp. are bacteria that cause persistent and latent infections. Treponema
palladium, the pathogen causing the disease syphilis is a sexually
transmitted disease which can have severe pathological consequences if
left untreated. The disease progresses through distinct stages. The
initial clinical stage is an ulcer or chancre at the site treponemal
inoculation. Following this is a period of spirochetemia and metastatic
distribution of microorganisms that continues, including repeating cycles
of infection and resolution in a condition known as secondary syphilis.
Following the resolution of secondary syphilis, the disease enters an
asymptomatic latency period which may conclude in tertiary syphilis,
which is a serious and often fatal condition. Tertiary syphilis may
manifest in (i) the heart as aortisis with aneurysis formation and
secondary aortic value insufficiency, (ii) central nervous system (tabes
dorsalis, general paresis), (iii) eyes (interstitial keratitis) or (iv)
ears (nerve deafness). Non-venereal forms resemble the clinical
manifestations of the venereal forms, but are transmitted primary by
direct contact and poor hygiene. They include yaws (T. pallidum subp.
pertenue,) pinta (T. carateum) and bejel (T. pallidum subsp. endemicum).

[0448] Treatments for syphilis include penicillin (E.g., penicillin G.),
tetracycline, doxycycline, ceftriaxone and azithromycin. The anti-PD-L1
antibodies of the invention would be most advantaneously administered to
treat the latent infection period.

[0449] Lyme disease, caused by Borrelia burgdorferi is transmitted into
humans through tick bites. The disease manifests initially as a localized
rash, followed by flu-like symptoms including malaise, fever, headache,
stiff neck and arthralgias. Later manifestations can include migratory
and polyarticular arthritis, neurologic and cardiac involvement with
cranial nerve palsies and radiculopathy, myocarditis and arrhythmias.
Some cases of Lyme disease become persistent, resulting in irreversible
damage analogous to tertiary syphilis.

[0450] Current therapy for Lyme disease includes primarily the
administration of antibiotics. Antibiotic-resistant strains may be
treated with hydroxychloroquine or methotrexate. Antibiotic refractory
patients with neuropathic pain can be treated with gabapentin.
Minocycline may be helpful in late/chronic Lyme disease with neurological
or other inflammatory manifestations. The anti-PD-L1 antibodies would be
most advantaneously administered to treat the latent infection period.

[0453] For infections resulting from viral causes, the anti-PD-L1
antibodies of the invention may be combined by application simultaneous
with, prior to or subsequent to application of standard therapies for
treating viral infections. Such standard therapies vary depending upon
type of virus, although in almost all cases, administration of human
serum containing antibodies (e.g., IgA, IgG) specific to the virus can be
effective.

[0454] 1) Influenza

[0455] Influenza infection results in fever, cough, myalgia, headache and
malaise, which often occur in seasonal epidemics. Influenza is also
associated with a number of postinfectious disorders, such as
encephalitis, myopericarditis, Goodpasture's syndrome, and Reye's
syndrome. Influenza infection also suppresses normal pulmonary
antibacterial defenses, such that patient's recovering from influenza
have an increased risk of developing bacterial pneumonia.

[0456] Influenza viral surface proteins show marked antigenic variation,
resulting from mutation and recombination. Thus, cytolytic T lymphocytes
are the host's primary vehicle for the elimination of virus after
infection. Influenza is classified into three primary types: A, B and C.
Influenza A is unique in that it infects both humans and many other
animals (e.g., pigs, horses, birds and seals) and is the principal cause
of pandemic influenza. Also, when a cell is infected by two different
influenza A strains, the segmented RNA genomes of two parental virus
types mix during replication to create a hybrid replicant, resulting in
new epidemic strains. Influenza B does not replicate in animals and thus
has less genetic variation and influenza C has only a single serotype.

[0457] Most conventional therapies are palliatives of the symptoms
resulting from infection, while the host's immune response actually
clears the disease. However, certain strains (e.g., influenza A) can
cause more serious illness and death. Influenza A may be treated both
clinically and prophylactically by the administration of the cyclic
amines inhibitors amantadine and rimantadine, which inhibit viral
replication. However, the clinical utility of these drugs is limited due
to the relatively high incidence of adverse reactions, their narrow
anti-viral spectrum (influenza A only), and the propensity of the virus
to become resistant. The administration of serum IgG antibody to the
major influenza surface proteins, hemagglutinin and neuraminidase can
prevent pulmonary infection, whereas mucosal IgA is required to prevent
infection of the upper respiratory tract and trachea. The most effective
current treatment for influenza is vaccination with the administration of
virus inactivated with formalin or β-propiolactone.

[0458] 2) Measles Virus

[0459] After an incubation of 9-11 days, hosts infected with the measles
virus develope fever, cough, coryza and conjunctivitis. Within 1-2 days,
an erythematous, maculopapular rash develop, which quickly spreads over
the entire body. Because infection also suppresses cellular immunity, the
host is at greater risk for developing bacterial superinfections,
including otitis media, pneumonia and postinfectious encephalomyelitis.
Acute infection is associated with significant morbidity and mortality,
especially in malnourished adolescents.

[0460] Treatment for measles includes the passive administration of pooled
human IgG, which can prevent infection in non-immune subjects, even if
given up to one week after exposure. However, prior immunization with
live, attenuated virus is the most effective treatment and prevents
disease in more than 95% of those immunized. As there is one serotype of
this virus, a single immunization or infection typically results in
protection for life from subsequent infection.

[0461] In a small proportion of infected hosts, measles can develop into
SSPE, which is a chronic progressive neurologic disorder resulting from a
persistent infection of the central nervous system. SSPE is caused by
clonal variants of measles virus with defects that interfere with virion
assembly and budding. For these patients, reactivation of T-cells with
the anti-PD-L1 antibodies of the invention so as to facilitate viral
clearance would be desirable.

[0462] 3) Hepatitis B Virus

[0463] Hepatitis B virus (HB-V) is the most infectious known bloodborne
pathogen. It is a major cause of acute and chronic heptatis and hepatic
carcinoma, as well as life-long, chronic infection. Following infection,
the virus replicates in hepatocytes, which also then shed the surface
antigen HBsAg. The detection of excessive levels of HBsAg in serum is
used a standard method for diagnosing a hepatitis B infection. An acute
infection may resolve or it can develop into a chronic persistent
infection.

[0464] Current treatments for chronic HBV include α-interferon,
which increases the expression of class I human leukocyte antigen (HLA)
on the surface of hepatocytes, thereby facilitating their recognition by
cytotoxic T lymphocytes. Additionally, the nucleoside analogs
ganciclovir, famciclovir and lamivudine have also shown some efficacy in
the treatment of HBV infection in clinical trials. Additional treatments
for HBV include pegylated a-interferon, adenfovir, entecavir and
telbivudine. While passive immunity can be conferred through parental
administration of anti-HBsAg serum antibodies, vaccination with
inactivated or recombinant HBsAg also confers resistance to infection.
The anti-PD-L1 antibodies of the invention may be combined with
conventional treatments for hepatitis B infections for therapeutic
advantage.

[0465] 4) Hepatitis C Virus

[0466] Hepatitis C virus (HC-V) infection may lead to a chronic form of
hepatitis, resulting in cirrosis. While symptoms are similar to
infections resulting from Hepatitis B, in distinct contrast to HB-V,
infected hosts can be asymptomatic for 10-20 years. Treatment for HC-V
infection includes the administration of a combination of
α-interferon and ribavirin. A promising potential therapy for HC-V
infection is the protease inhibitor telaprevir (VX-960). Additional
treatments include: anti-PD-1 antibody (MDX-1106, Medarex), bavituximab
(an antibody that binds anionic phospholipid phosphatidylserine in a
B2-glycoprotein I dependent manner, Peregrine Pharmaceuticals), anti-HPV
viral coat protein E2 antibod(y)(ies) (E.g., ATL 6865-Ab68+Ab65, XTL
Pharmaceuticals) and Civacir® (polyclonal anti-HCV human immune
globulin). The anti-PD-L1 antibodies of the invention may be combined
with one or more of these treatments for hepatitis C infections for
therapeutic advantage.

[0467] Protease, polymerase and NS5A inhibitors which may be used in
combination with the anti-PD-L1 antibodies of the invention to
specifically treat Hepatitis C infection include the following identified
in Table B

[0469] HIV attacks CD4+ cells, including T-lymphocytes,
monocyte-macrophages, follicular dendritic cells and Langerhan's cells,
and CD4+ helper/inducer cells are depleted. As a result, the host
acquires a severe defect in cell-mediated immunity. Infection with HIV
results in AIDS in at least 50% of individuals, and is transmitted via
sexual contact, administration of infected blood or blood products,
artificial insemination with infected semen, exposure to blood-containing
needles or syringes and transmission from an infected mother to infant
during childbirth.

[0471] Treatments for HIV include antiviral therapies including nucleoside
analogs, zidovudine (AST) either alone or in combination with didanosine
or zalcitabine, dideoxyinosine, dideoxycytidine, lamidvudine, stavudine;
reverse transcriptive inhibitors such as delavirdine, nevirapine,
loviride, and proteinase inhibitors such as saquinavir, ritonavir,
indinavir and nelfinavir. The anti-PD-L1 antibodies of the invention may
be combined with conventional treatments for HIV infections for
therapeutic advantage.

[0472] 6) Cytomegalovirus

[0473] Cytomegalovirus (CMV) infection is often associated with
persistent, latent and recurrent infection. CMV infects and remains
latent in monocytes and granulocyte-monocyte progenitor cells. The
clinical symptoms of CMV include mononucleosis-like symptoms (i.e.,
fever, swollen glands, malaise), and a tendancy to develop allergic skin
rashes to antibiotics. The virus is spread by direct contact. The virus
is shed in the urine, saliva, semen and to a lesser extent in other body
fluids. Transmission can also occur from an infected mother to her fetus
or newborn and by blood transfusion and organ transplants. CMV infection
results in general impairment of cellular immunity, characterized by
impaired blastogenic responses to nonspecific mitogens and specific CMV
antigens, diminished cytotoxic ability and elevation of CD8 lymphocyte
number of CD4+ lymphocytes.

[0474] Treatments of CMV infection include the anti-virals ganciclovir,
foscarnet and cidovir, but these druges are typically only prescribed in
immunocompromised patients. The anti-PD-L1 antibodies of the invention
may be combined with conventional treatments for cytomegalovirus
infections for therapeutic advantage.

[0475] 7) Epstein-Barr Virus

[0476] Epstein-Barr virus (EBV) can establish persistent and latent
infections and primarily attacks B cells. Infection with EBV results in
the clinical condition of infectious mononucleosis, which includes fever,
sore throat, often with exudate, generalized lymphadenopathy and
splenomegaly. Hepatitis is also present, which can develop into jaundice.

[0477] While typical treatments for EBV infections are palliative of
symptoms, EBV is associated with the development of certain cancers such
as Burkitt's lymphoma and nasopharyngeal cancer. Thus, clearance of viral
infection before these complications result would be of great benefit.
The anti-PD-L1 antibodies of the invention may be combined with
conventional treatments for Epstein-Barr virus infections for therapeutic
advantage.

[0478] 8) Herpes Virus

[0479] Herpes simplex virus (HSV) is transmitted by direct contact with an
infected host. A direct infection may be asymptomatic, but typically
result in blisters containing infectious particles. The disease manifests
as cycles of active periods of disease, in which lesions appear and
disappear as the viral latently infect the nerve ganglion for subsequent
outbreaks. Lesions may be on the face, genitals, eyes and/or hands. In
some case, an infection can also cause encephalitis.

[0480] Treatments for herpes infections are directed primarily to
resolving the symptomatic outbreaks, and include systemic antiviral
medicines such as: acyclovir (e.g., Zovirax®), valaciclovir,
famciclovir, penciclovir, and topical medications such as docosanol
(Abreva®), tromantadine and zilactin. The clearance of latent
infections of herpes would be of great clinical benefit. The anti-PD-L1
antibodies of the invention may be combined with conventional treatments
for herpes virus infections for therapeutic advantage.

[0481] 9) HTLV

[0482] Human T-lymphotrophic virus (HTLV-1, HTLV-2) is transmitted via
sexual contact, breast feeding or exposure to contaminated blood. The
virus activates a subset of TH cells called Th1 cells, resulting in
their overproliferation and overproduction of Th1 related cytokines
(e.g., IFN-γ and TNF-α). This in turn results in a
suppression of Th2 lymphocytes and reduction of Th2 cytokine production
(e.g., IL-4, IL-5, IL-10 and IL-13), causing a reduction in the ability
of an infected host to mount an adequate immune response to invading
organisms requiring a Th2-dependent response for clearnance (e.g.,
parasitic infections, production of mucosal and humoral antibodies).

[0483] HTLV infections cause lead to opportunistic infections resulting in
bronchiectasis, dermatitis and superinfections with Staphylococcus spp.
and Strongyloides spp. resulting in death from polymicrobial sepsis. HTLV
infection can also lead directly to adult T-cell leukemia/lymphoma and
progressive demyelinating upper motor neuron disease known as HAM/TSP.
The clearance of HTLV latent infections would be of great clinical
benefit. The anti-PD-L1 antibodies of the invention may be combined with
conventional treatments for HTLV infections for therapeutic advantage.

[0484] 10) HPV

[0485] Human papilloma virus (HPV) primarily affects keratinocytes and
occurs in two forms: cutaneous and genital. Transmission it believed to
occurs through direct contact and/or sexual activity. Both cutaneous and
genital HPV infection, can result in warts and latent infections and
sometimes recurring infections, which are controlled by host immunity
which controls the symptoms and blocks the appearance of warts, but
leaves the host capable of transmitting the infection to others.

[0486] Infection with HPV can also lead to certain cancers, such as
cervical, anal, vulvar, penile and oropharynial cancer. There are no
known cures for HPV infection, but current treatment is topical
application of Imiquimod, which stimulates the immune system to attack
the affected area. The clearance of HPV latent infections would be of
great clinical benefit. The anti-PD-L1 antibodies of the invention may be
combined with conventional treatments for HPV infections for therapeutic
advantage.

[0487] c. Fungus

[0488] Fungal infections, or mycoses, can result as either a primary
infection or as opportunistic colonization of hosts with compromised
immune systems by endogenous flora. Immunity to mycoses is principally
cellular, involving neutrophils, macrophages, lymphocytes and probably
natural killer (NK) cells. Mycoses are typically not susceptible to
direct killing by antibody and complement. Systemic invasive mycoses
resulting from primary infection include blastomycosis,
coccidioiodomycosis, histoplamosis, and paracoccidioiodmycosis. For
chronic infections results from fungal infections, the anti-PD-L1
antibodies of the invention may be administered prior to simultaneous
with or subsequent to any of the conventionally known treatments for
these mycoses.

[0489] Blastomycosis, caused by Blastomyces dermatitis is
inhalation-acquired and produces a primary pulmonary infection or
hematogenously disseminated disease involving predominantly skin, bones,
and the male genitourinary tract. Primary exposure may be asymptomatic,
or it may produce an influenza-like syndrome. This disease can manifest
in a chronic indolent form. The disease is also associated with
compromised immune such as in patients with AIDS. Conventional therapy
for B. dermatitis infection include itraconazole, ketoconazole or
intravenous injection of amphotericin B.

[0490] Coccidioiodmycosis, caused by Coccidioides immitis, is
inhalation-acquired and can cause primary pulmonary infection,
progressive pulmonary disease, or hematogenously disseminated disease
involving predominantly skin, subcutaneous tissues, bones, joints, and
meninges. Primary exposure may be asymptomatic (60%) or associated with
an influenza-like syndrome. Pneumonia, pleuritis, and pulmonary
cavitation may occur. Metastatic manisfestations include skin lesions,
including nodules, ulcers, sinus tracts from deeper loci and verrucouse
granulomas, bones, joints, tendon sheaths and meninges, including
meningitis. The disease is also associated with compromised immunity such
as in patients with AIDS. Treatment for coccidioidiomycosis includes
ketoconazole, intraconazole and fluconazole, especially for long-term
maintenance therapy of nonmeningial disease. Meningial forms are treated
usually with intrathecal administration of Amphotericin B.

[0491] Histoplasmosis, caused by Histoplasma capsulatum, is an
inhalation-acquired disease of the reticuloendothelial system in which
tiny yeasts reside in macrophages. It can produce primary pulmonary
infection, progressive pulmonary disease or hematogenously disseminated
disease involving predominantly the reticuloendothelial system, mucosal
surfaces, and adrenal glands. Reactivation of latent infections often
occur in patients with compromised immunity, such as in patients with
AIDS. Primary exposure may be asymptomatic or associated with a flu-like
syndrome, including pneumonia, pleuritis, pulmonary cavitation and
mediastinal adenopathy. Metastatic sites include the reticuloendothelial
system (hepatosplenomegaly, lymphadenopathy, anemia, leucopenia and
thrombocytopenia), mucous membranes (oronasopharnygeal ulcerations),
gastrointestinal tract (malabsorption), and adrenal insufficiency. While
most primary infections resolve spontaneously, when associated with
compromised immunity such as in patients with AIDS, relapse is ongoing
and is often associated with hematogenous pneumonia, ARDS, disseminated
intravascular coagulation (DIC), hematogenously distributed
papulopustules and meningitis. Histoplasmosis is treated with
Amphotericin B (especially in immunocompromised patients acutely ill with
hematogenous dissemination), intraconzoles and ketoconazole.

[0492] Paracoccidioiomycosis, caused by Paracoccidioides brasiliensis, is
an inhalation-acquired mycosis that can produce primary pulmonary
infection or hematogenously disseminated disease involving predominantly
the skin, mucouse membranes, reticulendothelial system and adrenals.
Infection may be initially asymptomatic but dormant, and then revive.
Treatment of this infection uses ketoconazole, intraconzole and
sulfonamides.

[0493] Systemic invasive mycoses resulting from opportunistic pathogens,
which occur in immunocompromised hosts, include candidiasis,
cryptococcosis, aspergillossi, mucomycosis and pneumocystosis. By
heightening immune response in a compromised immune system, the
anti-PD-L1 antibodies of the invention may also have therapeutic value in
the treatment of these conditions, especially when combined with
conventional therapies.

[0494] Treatments for candidiasis (caused by Candida albicans, C.
tropicalis, C. glabrata), crytococcosis (caused by Cryptococcus
neoformans), aspergillosis (caused by Aspergillus flavus, A. fumigatus,
A. tereus and A. niger) and mucormycosis (caused by Rhizopus arrhizus,
Rhizomuco, Absidia, Cunninghamella, Mortierella, Saksenaea spp.) may be
treated by one or more of the following imidazole, ketoconazole,
intraconazole, fluconazole, amphotericin B with and without flucytosine.
Pneumocystitis (caused by penumocystis carnii) recently reclassified from
protozoan to fungi is treated with trimethoprim-sulfamethoxole (TMP-SMZ)
and intravenous pentamidine isethionate, as well as dapsone, TMP-dapson,
trimetrexate, clindamycin-primaquine and atovagnone.

[0496] Infections are believed to be transmitted to humans from direct
contact with animals, contaminated water or another infected host. After
infecting host cells, the sporoplasm grows, dividing or forming a
multinucleate plasmodium which can have complex life cycles including
both asexual and sexual reproduction. Autoinfection by successive
generations and chronic, debilitating diseases often characterize
Microsporidial infections.

[0497] The clinical manifestations of the disease can vary depending on
the species and the host's immune status, and include conjunctivitis
(e.g., V. corneae), chronic diarrhea, malabsorption and wasting (e.g., E.
bieneusi, E. intestinalis).

[0498] Treatments for ocular, intestinal and disseminated microsporosis
includes administration of albendazole. Topical application of fumagillin
may also be used effectively to treat microsporidial
keratoconjunctivitis. Other drugs include antihelminthics (e.g.,
albendazole), antibiotics (e.g., fumagillin), immunomodulators (e.g.,
thalidomide), antiprotozoals, (e.g., metronidazole).

[0499] d. Protozoan

[0500] Disease resulting from parasitic disorders such as malaria,
schistosomiasis and leishmaniasis are among the most prevalent and
important health problems in developing countries. These diseases pose
particular challenges because they may evade host immunity through
various means, including: 1) living inside host cells (e.g., Leishmania),
2) rapidly change surface antigens (e.g., trypansomes) and 3)
"disguising" themselves as host cells by displaying host antigens (e.g.,
schistosomisasis). The use of immunosuppressive drugs in the treatment of
cancer and in conjunction with organ transplants, as well the global
prevalence of AIDs can reactivate latent or subclinical infections from
Plasmodium spp., Toxoplasma spp., Leishmania spp., Cryptosporidium spp.,
Trypanosoma spp. and helminths.

[0501] For chronic infections resulting from infections with protozoan
parasites, the anti-PD-L1 antibodies of the invention may be combined by
administration in combination with, prior to or subsequent with standard
anti-protozoan therapies.

[0502] Malaria, caused by parasites of genus Plasmodium (E.g., P. ovale,
P. malariae, P. falciparum, P. vivax), begins the infectious cycle as a
sporozite which developes in the gut of the female anopheline mosquito.
Upon transmission into humans, these sporozites invade and multiply
within hepatic cells without inducing an inflammatory reaction. The
progeny of these organisms, called merozoites, then invade erythrocytic
cells and initiate the clinical phase of the disease, typically
characterized by fever and chills. In areas of the world where infection
is endemic, nearly all residents harbor continuous low level chronic
infections of low to moderate pathogenicity, with increasing levels of
IgG antibodies providing protection from merozoite entry into
erythrocytes.

[0504] Through reactivating anerigic T-cells, the anti-PD-L1 antibodies of
the invention may particularly therapeutic in aiding clearance of
malarial parasites.

[0505] Toxoplasmosis, caused by parasites of the genus Toxoplasma, is
often asymptomatic, but a small fraction can develop clinical disease,
which can range from benign lymphadenopathy acute to fatal infections of
the central nervous system. The sources of infection include cysts in raw
or partially cooked pork or mutton and oocytes passed in feces of
infected cats. Infection occus in humans usually through the
gastrointestinal tract, and the protozoa can penetrate and proliferate
(as tachyzoites) in virtually every cell of the body. These tachyzoites
can produce cysts filled with minute slow-growing infective bodies
(bradyzoites) that remain viable for long periods of time, resulting in a
latent chronic infection. Hosts with compromised immune systems, such as
those taking immunosuppressive drugs or suffering from HIV are
particularly prone to suffering from toxicoplasmosis.

[0506] Medications that are used to treat primary toxoplasmosis include
the following: pyrimethamine, both with and without an accompanying
antibiotics (E.g., sulfadiazine, clindamycin, spiramycin and
minocycline). Latent toxoplasmosis may be treated with the antibiotics
atovaquone, both with and without clindamycin.

[0507] Leishmaniasis, caused by parasites of the genus Leishmania, infect
macrophages of the skin and viscera, and is transmitted into humans
through sandflies. As there is little or no specific serum antibody,
cell-mediation immunity through activated T-cells appears to be a
critical route by which infection is cleared. Old World Leishmaniasis,
also known as tropical sore, is caused by several species of Leishmania:
L. tropica, L. major and L. aethiopica. New World Leishmaniasis is caused
by various subspecies of L. Mexicana and L. braziliensis. These parasites
induce a strong cell-mediated immune response, but the outcome of the
clinical disease results also in part to the host response. If the host
mounts in a suppressed or inadequate cell-mediated response, the result
is diffuse chronic cutaneous leishmaniasis, with little hope for
spontaneous cure (E.g. L. aethiopica, L. Mexicana). If the host mounts an
excessive cell-mediated response, the response is a lupoid or recidiva
leishmaniasis, with persistent nonulcerated lymphoid nodules appearing at
the edge of primary lesions (E.g., L. tropica). Recidiva leishmaniasis
can appear from 1 to 10 years after the initial lesion. There are two
forms of the disease, cutaneous and visceral, with the cutaneous form
manifesting in cutaneous lesions with cell mediated immunity is critical
to clearance. In the visceral form, cell-mediated immunity is
insufficient or non-existent, and the disease manifest clinically as
polyclonal B-cell hypergammaglobulinemia, leukopenia, splenomegaly and
elevated production of TNF-α.

[0508] Miltefosine (E.g., Impavido®) and paramyocin are currently
available treatments for both cutaneous and visceral leishmaniasis.

[0509] Crytosporidiosis, caused by infections from protozoans of the genus
Crytosporidia and results from direct human contact with fecal excrement
of infected hosts. The infection of intestinal mucosal tissue can result
in diarrhea. The disease typically manifests as an acute infection, but
it can become chronic, especially in immunocompromised individuals.
Treatments are typically palliative, especially hydration, but
paromomycin, azithromycin and serum Ig (e.g., Lactobin-R®) have been
successful in clearing infection.

[0510] Trypanosomiasis, caused by the parasite Trypanosoma (E.g., T.
Brucei, subsp. gambiense, rodesiense infects humans and cattle through
bites from the Tsetse-fly. The challenge that this pathogen poses results
from successive generations of populations with displaying different
surface antigens. Infections are characterized by elevated levels of
non-specific and non-protective serum immunoglobulins.

[0511] Treatments for Trypanosomiasis include intravenous administration
of the following: pentamidine (for T.b. gambiense), intravenous suramin
(for T.b. rhodesiense), eflornithine, melarsoprol both with and without
nifurtimox.

[0513] Schistosomiasis (aka bilharzia), caused by Shistosoma mansoni, S.
japonicum, S. haematobium and S. mekongi start their life cyle as eggs in
water, which then hatch into miracidia, which penetrate snails and create
multiple generations of sporocysts. These in turn produce fork-tailed
cercariae which can infect the bloodstream of a human host as a
shistosomula, which migrate initially to the lungs, and then to the
liver. These flukes eventually pair, mate, and lay eggs in the mesenteric
venules. While many of these eggs travels to the intestines and are
excreted, some are trapped in the submucosa, portal venules of the liver
and other organs of the body. The granulomatous inflammation associated
with the trapped eggs is the definitive symptom of chronic
schistomsomiasis.

[0515] Cestode infections can be classified into two groups, one is the
intestinal dwelling adult tapeworms such as Diphyllobothrium latum and
Taenia saginata, which have a restricted, non-humoral immune effect. The
second group describes a migratory tissue-encysting larval tapeworms such
as Hymenolepis nana, Echinococcus granulosus and Taenia solium, which
induce strong parenteral host responses and protective serum antibodies.
The most serious cestode infection in human is Echinococcosis, which when
implanted in the liver, lungs, brain, kidneys or other parts of the body
can result in the formation of hydatid cysts.

[0516] Treatments for Echinococcosis include administration of
metronidazole, albendazole and surgical intervention, such as removal,
aspiration, marsupialization or omentopexy.

[0517] Nematodes are the most common varied and widely distributed
helminths that infect humans, caused disorders such as trichinosis,
ascariasis, filariosis and strongylodiosis. Trichinosis, caused by
Trichinella spiralis, can result from ingestion of the larvae of T.
spiralis in raw meat or partially cooked meat such as pork. In humans,
infections elicit strong humoral response with elevated IgM, followed by
IgG production, followed by rapid expulsion of antibody-damaged worms by
T-lymphocytes.

[0518] The only known treatment for killing adult worms in the intestine
is thiabendazole, while there is no known treatment to kill the larvae.

[0519] Ascaris, also known as giant roundworm (Ascaris lumbricoides), is a
common parasite in humans resulting from ingestion of
fecally-contaminated substances. While patients can remain asymptomatic
for very long periods of time, as larval stages travel through the body,
they may cause visceral damage, peritonitis and inflammation, enlargement
of the liver or spleen, toxicity, and pneumonia.

[0520] Treatments for ascariasis include administration of mebendazole
(E.g., Vermox®), piperazine, pyrantel pamoate (E.g., Antiminth®,
Pin-Rid®, Pin-X®), albendazole, thiabendazole with or without
piperazine, hexylresorcinol, santonin and oil of Chenopodium. The
anti-PD-L1 antibodies of the invention may be administered in combination
with, prior or subsequent to administration of these therapies for the
treatment of ascariasis.

[0521] Filariosis, caused by filarid nematodes, are introduced into humans
by insect vectors. Onchocerca volvulus, which caused onchoceriasis or
river blindness, is transmitted by bites from the blackfly. Infectious
larvae lodge themselves subcutaneously and develop into adults, induce a
fibrogenic host response, and shed large amount of microfilariae, which
disperse subcutaneously and throughout the eyes, further inducing a
keratisis or retininitis which then causes the cornea to become opaque.
Lymphatic filariasis results from infection by Brugia spp. and Wuchereria
spp. Over time, scarring of the lymph tissue, especially in the groin,
may prevent draining, resulting in the disfiguring condition
elephantiasis.

[0522] The primary treatment for filariosis is the administration of the
antibiotic ivermectin, abendazole and diethylcarbamazine citrate (DEC,
Hetrazan®) with or without ivermectin or albendazole. Other treatment
prospects includes doxycycline, which kills a symbiotic bacteria,
wolbochia.

[0523] Strongylodiosis, caused by parasites of the genus Strongyloides
(E.g., S. stercoralis, S. fulleborni), is a disease that is passed to
humans through fecally contaminated soil. They can exist in both a free
living cycle (rabditiform larvae maturing into adult worms) as well as a
parasitic cycle (filariform larvae maturing into adult worms) which
penetrates the skin, travel to the lungs, then the pharynx and ultimately
reside in the intestine. Autoinfection with Strongyloides is also known
to occur, which is essentially repeated infection by successive
generations of filariform larvae.

[0524] Infections may be aymptomatic, or can be characterized by pain and
diarrhea in the gastrointestinal tract, Loffler's syndrome in the lungs
(i.e., eosinophila) and urticaria. Blood eosinophila may also be present.
As persistent infection of Strongyloides can mimic peptic ulcer,
gallbladder disease and Crohn's disease, misdiagnosis is common. It is a
particular problem in immunocompromised hosts.

[0525] Known treatments for Strongyloidiosis is ivermectin, albenazole or
thiabendazole but as this mediation only kills adult worms, repeated
administration is necessary.

[0526] e. Vaccination

[0527] Vaccination or the administration of antigenic material to induce
immunity to disease is routinely used to prevent or ameliorate the
effects of infection by a pathogen Enhancing host immunity can be used on
undesired antigens found not only on infectious pathogens, but also host
tissue that has become diseased (e.g., cancerous). Traditionally,
vaccines are derived from weakened or dead whole pathogens, but they can
also be peptides representing epitopes on the intact pathogen that are
specifically recognized by human class I or class II major
histocompatability complex (MHC) molecules. Peptide antigens of
particular interest are those which are specifically recognized by T
cells.

[0528] Recently, it has been shown that combining a therapeutic
vaccination with administration of PD-L1 blockade on exhausted CD8+ T
cells resulted in enhanced function and viral control in a chronic
infection mouse model. Ha et al., J. Exp. Med. 205(3): 543-555 (2008). As
a result, the anti-PD-L1 antibodies described herein may also be combined
with antigen vaccination (e.g., administered prior, simultaneous or
after) to treat infection (e.g., acute and chronic) resulting from viral,
bacterial, fungal or protozoan invasion as well as tumor immunity.

[0529] G. Pharmaceutical Dosages:

[0530] Dosages and desired drug concentration of pharmaceutical
compositions of the present invention may vary depending on the
particular use envisioned. The determination of the appropriate dosage or
route of administration is well within the skill of an ordinary artisan.
Animal experiments provide reliable guidance for the determination of
effective doses for human therapy. Interspecies scaling of effective
doses can be performed following the principles laid down by Mordenti, J.
and Chappell, W. "The Use of Interspecies Scaling in Toxicokinetics," In
Toxicokinetics and New Drug Development, Yacobi et al., Eds, Pergamon
Press, New York 1989, pp. 42-46.

[0531] When in vivo administration of the polypeptides or antibodies
described herein are used, normal dosage amounts may vary from about 10
ng/kg up to about 100 mg/kg of mammal body weight or more per day,
preferably about 1 mg/kg/day to 10 mg/kg/day, depending upon the route of
administration. Guidance as to particular dosages and methods of delivery
is provided in the literature; see, for example, U.S. Pat. No. 4,657,760;
5,206,344; or 5,225,212. It is within the scope of the invention that
different formulations will be effective for different treatments and
different disorders, and that administration intended to treat a specific
organ or tissue may necessitate delivery in a manner different from that
to another organ or tissue. Moreover, dosages may be administered by one
or more separate administrations, or by continuous infusion. For repeated
administrations over several days or longer, depending on the condition,
the treatment is sustained until a desired suppression of disease
symptoms occurs. However, other dosage regimens may be useful. The
progress of this therapy is easily monitored by conventional techniques
and assays.

[0532] H. Administration of the Formulation

[0533] The formulations of the present invention, including but not
limited to reconstituted and liquid formulations, are administered to a
mammal in need of treatment with the anti-PD-L1 antibodies, preferably a
human, in accord with known methods, such as intravenous administration
as a bolus or by continuous infusion over a period of time, by
intramuscular, intraperitoneal, intracerobrospinal, subcutaneous,
intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation
routes.

[0534] In preferred embodiments, the formulations are administered to the
mammal by subcutaneous (i.e. beneath the skin) administration. For such
purposes, the formulation may be injected using a syringe. However, other
devices for administration of the formulation are available such as
injection devices (e.g. the INJECT-EASE® and GENJECT® devices);
injector pens (such as the GENPEN®); auto-injector devices, needleless
devices (e.g. MEDIJECTOR® and BIOJECTOR®); and subcutaneous patch
delivery systems.

[0535] In a specific embodiment, the present invention is directed to kits
for a single dose-administration unit. Such kits comprise a container of
an aqueous formulation of therapeutic protein or antibody, including both
single or multi-chambered pre-filled syringes. Exemplary pre-filled
syringes are available from Vetter GmbH, Ravensburg, Germany.

[0536] The appropriate dosage ("therapeutically effective amount") of the
protein will depend, for example, on the condition to be treated, the
severity and course of the condition, whether the protein is administered
for preventive or therapeutic purposes, previous therapy, the patient's
clinical history and response to anti-PD-L1 antibody, the format of the
formulation used, and the discretion of the attending physician. The
anti-PD-L1 antibody is suitably administered to the patient at one time
or over a series of treatments and may be administered to the patient at
any time from diagnosis onwards. The anti-PD-L1 antibody may be
administered as the sole treatment or in conjunction with other drugs or
therapies useful in treating the condition in question.

[0537] For anti-PD-L1 antibodies, an initial candidate dosage can range
from about 0.1-20 mg/kg for administration to the patient, which can take
the form of one or more separate administrations. However, other dosage
regimens may be useful. The progress of such therapy is easily monitored
by conventional techniques.

[0538] I. Articles of Manufacture

[0539] In another embodiment of the invention, an article of manufacture
is provided which contains the formulation and preferably provides
instructions for its use. The article of manufacture comprises a
container. Suitable containers include, for example, bottles, vials (e.g.
dual chamber vials), syringes (such as single or dual chamber syringes)
and test tubes. The container may be formed from a variety of materials
such as glass or plastic. The container holds the formulation. The label,
which is on, or associated with the container may indicate directions for
reconstitution and/or use. The label may further indicate that the
formulation is useful or intended for subcutaneous administration, and/or
for the treatment of a T-cell dysfunctional disorder. The container
holding the formulation may be a multi-use vial, which allows for repeat
administrations (e.g. from 2-6 administrations) of the reconstituted
formulation. The article of manufacture may further comprise a second
container comprising a suitable diluent (e.g. BWFI). Upon mixing of the
diluent and the lyophilized formulation, the final protein concentration
in the reconstituted formulation will generally be at least 50 mg/ml. The
article of manufacture may further include other materials desirable from
a commercial and user standpoint, including other buffers, diluents,
filters, needles, syringes, and package inserts with instructions for
use.

[0540] The invention will be more fully understood by reference to the
following examples. They should not, however, be construed as limiting
the scope of the invention. All citations throughout the disclosure are
hereby expressly incorporated by reference.

[0541] In another embodiment, the invention provides for an article of
manufacture comprising the formulations described herein for
administration in an auto-injector device. An auto-injector can be
described as an injection device that upon activation, will deliver its
contents without additional necessary action from the patient or
administrator. They are particularly suited for self-medication of
therapeutic formulations when the delivery rate must be constant and the
time of delivery is greater than a few moments.

[0543] After 4 rounds of panning, significant enrichment was observed. 96
clones were picked each from VH and VH/VL library sorting to determine
whether they specifically bound to both human and murine PD-L1-Fc. The
variable regions of these clones were PCR sequenced to identify unique
sequence clones.

[0544] The parental clones of interest were reformatted into IgGs by
cloning VL and VH regions of individual clones into the LPG3
and LPG4 vector (Lee et al., supra), respectively, transiently expressing
in mammalian CHO cells, and purifying with a protein A column. The 13
Phage antibodies were evaluated for their ability to block the
interaction between soluble PD-1-Fc fusion protein and human or mouse
PD-L1 expressed in 293 cells (IC 50 values are designated in Table
1--upper half). YW243.55, the antibody with the lowest IC50 for
blocking human PD-L1 binding to PD-1 was selected for subsequent affinity
maturation to improve its affinity for both human and mouse PD-L1. (Table
1). An antibody with comparable cross reactivity against both primate and
murine species (as well as retaining affinity to human) would provide for
a therapeutic of enhanced value, in that the same antibody that has been
well characterized in experimental models can be used in human clinical
trials. This avoids the uncertainly resulting from the use of a
model-specific surrogate.

Construct Libraries for Affinity Improvement of Clones Derived from the
VH Library

[0545] Phagemid pW0703 (derived from phagemid pV0350-2b (Lee et al., J.
Mol. Biol 340: 1073-1093 (2004)), containing stop codon (TAA) in all
CDR-L3 positions and displaying monovalent Fab on the surface of M13
bacteriophage) served as the library template for grafting heavy chain
variable domains (VH) of clones of interest from the VH library
for affinity maturation. Both hard and soft randomization strategies were
used for affinity maturation. For hard randomization, one light chain
library with selected positions of the three light chain CDRs was
randomized using amino acids designed to mimic natural human antibodies
and the designed DNA degeneracy was as described in Lee et al. (J. Mol.
Biol 340, 1073-1093 (2004)). For soft randomization, residues at
positions 91-94, and 96 of CDR-L3, 28-31 and 34-35 of CDR-H1, 50, 52, and
53-58 of CDR-H2, 95-99 and 100 A of CDR-H3, were targeted; and two
different combinations of CDR loops, L3/H1/H2 and L3/H3, were selected
for randomization. To achieve the soft randomization conditions, which
introduced the mutation rate of approximately 50% at the selected
positions, the mutagenic DNA was synthesized with 70-10-10-10 mixtures of
bases favoring the wild type nucleotides (Gallop et al., Journal of
Medicinal Chemistry 37:1233-1251 (1994)).

Phage Sorting to Generate Affinity Improvement

[0546] The phage clones previously identified were subjected to plate
sorting for the first round, followed by five or six rounds of solution
sorting. The libraries were sorted against human and murine PD-L1-Fc
separately (R&D Systems, cat. #156-B7, cat #1019-B7, respectively). For
the human PD-L1-Fc target, at the first round of plate sorting, three
libraries were sorted against target coated plate (NUNC Maxisorp®
plate) separately with phage input about 3 O.D./ml in 1% BSA and 0.05%
Tween 20 for 2 hours at room temperature. After the first round of plate
sorting, solution sorting was performed to increase the stringency of
selection. For solution sorting, 1 O.D./ml phage propagated from the
first round of plate sorting were incubated with 20 nM biotinylated
target protein (the concentration is based on parental clone phage
IC50 value) in 100 μL buffer containing 1% Superblock (Pierce
Biotechnology) and 0.05% Tween-20 for 30 minutes at room temperature. The
mixture was further diluted 10× with 1% Superblock, and 100
μL/well was applied to neutravidin-coated wells (5 μg/ml) for 15
minutes at room temperature with gentle shaking such that biotinylated
target bound phage. The wells were washed 10× with PBS-0.05%
Tween-20. To determine background binding, control wells containing phage
with targets that were not biotinylated were captured on
neutravidin-coated plates. Bound phage was eluted with 0.1N HCl for 20
minutes, neutralized by 1/10 volume of 1M Tris pH-11, titered, and
propagated for the next round. Next, five more rounds of solution sorting
were carried out together with two methods of increasing selection
stringency. The first of which is for on-rate selection by decreasing
biotinylated target protein concentration from 4 nM to 0.5 nM, and the
second of which is for off-rate selection by adding excess amounts of
non-biotinylated target protein (100˜2000 fold more) to compete off
weaker binders either at room temperature or 37° C. Also, the
phage input was decreased (0.1˜0.5 O.D/ml) to lower background
phage binding. For murine PD-L1-Fc target, phage sorting method is
similar to the one described above for the human PD-L1 Fc antigen, with a
few modifications. Specifically, 100 nM biotinylated murine PD-L1-Fc was
used for solution panning immediately after first round of plate panning.
In the four subsequent rounds of solution panning, biotinylated target
was reduced from 10 nM to 1 nM, and 200-500 fold excess of
non-biotinylated target was added at room temperature.

[0547] The affinity matured clones were then further screened with the
High Throughput Affinity Screening ELISA procedure described in the
following example.

High Throughput Affinity Screening ELISA (Single Spot Competition)

[0548] Colonies were picked from the seventh and sixth round screens for
the human and murine PD-L1 target, respectively. Colonies were grown
overnight at 37° C. in 150 μL/well of 2YT media with 50
μg/ml carbenicillin and 1E10/ml KO7 in 96-well plate (Falcon). From
the same plate, a colony of XL-1 infected parental phage was picked as
control. 96-well Nunc Maxisorp® plates were coated with 100
μL/well of human and murine PD-L1-Fc protein (2 μg/ml) separately
in PBS at 4° C. overnight or room temperature for 2 hours. The
plates were blocked with 65 μL of 1% BSA for 30 min and 40 μL of 1%
Tween 20 for another 30 minutes.

[0549] The phage supernatant was diluted 1:10 in ELISA (enzyme linked
immunosorbent assay) buffer (PBS with 0.5% BSA, 0.05% Tween-20) with or
without 10 nM target protein in 100 μL total volume and incubated at
least 1 hour at room temperature in an F plate (NUNC). 75 μL of
mixture with or without target protein was transferred side by side to
the target protein coated plates. The plate was gently shaken for 15 min
to allow the capture of unbound phage to the target protein-coated plate.
The plate was washed at least five times with PBS-0.05% Tween-20. The
binding was quantified by adding horseradish peroxidase (HRP)-conjugated
anti-M13 antibody in ELISA buffer (1:5000) and incubated for 30 minutes
at room temperature. The plates were washed with PBS-0.05% Tween 20 at
least five times. Next, 100 μL/well of a 1:1 ratio of
3,3',5,5'-tetramethylbenzidine (TMB) Peroxidase substrate and Peroxidase
Solution B (H2O2) (Kirkegaard-Perry Laboratories (Gaithersburg,
Md.)) was added to the well and incubated for 5 minutes at room
temperature. The reaction was stopped by adding 100 μL 1M Phosphoric
Acid (H3PO4) to each well and allowed to incubate for 5 minutes
at room temperature. The OD (optical density) of the yellow color in each
well was determined using a standard ELISA plate reader at 450 nm. The OD
reduction (%) was calculated by the following equation.

OD450nm reduction (%)=[(OD450nm of wells with
competitor)/(OD450nm of well with no competitor)]×100

[0550] In comparison to the OD450nm reduction (%) of the well of
parental phage (100%), clones that had the OD450nm reduction (%)
lower than 50% for both the human and murine target were picked for
sequence analysis. Unique clones were selected for phage preparation to
determine binding affinity (phage IC50) against both human and
murine PD-L-Fc by comparison with parental clones.

[0552] PD-1-Fc, and B7.1-Fc proteins were biotinylated with EZ-Link
sulfo-NHS-LC-LC-biotin (Pierce) for 30 minutes at room temperature as
described by the manufacturer. Excess non-reacted biotin was removed with
Quick Spin High Capacity Columns, G50-Sephadex (Roche) as described by
the manufacturer.

[0554] Antibody concentrations resulting in 50% inhibition (IC50) of
the binding of hPD-1-Fc to hPD-L1 expressing 293 cells were measured by
electrochemiluminescent (ECL) cell-binding assay. hPD-L1 expression 293
cells were washed with phosphate buffered saline (PBS) and seeded at
25,000 cells per well in 25 μL PBS on 96 well High Bind plate (Meso
Scale Discovery). Incubate plate at room temperature to allow the cells
to attach to the carbon surface of the plate. Add 25 μL of 30% FBS to
each well and incubate the plate for 30 minutes with mild agitation to
block non-specific binding sites. Wash plate three times with PBS on an
ELISA microplate washer (ELx405 Select, Bio-Tek Instruments) under gentle
dispense and aspiration conditions. Remove excess PBS in the wells by
blotting plate on paper towels. Add 12.5 μL of 2× concentration
of antibodies to each well in 3% FBS in PBS (Assay Buffer) and followed
by 12.5 μL of 4 μg/mL (2× concentration) of hPD-1-biotin in
Assay Buffer and incubate plate for one hour with mild agitation. Wash
plate 3× with PBS on a microplate washer, and blot plate on paper
towels. Add 25 μL of 2 μg/mL of Streptavidin-Ruthenium (Meso Scale
Discovery) and incubate in assay buffer at room temperature for 30
minutes with gentle agitation. Wash 3× with PBS on microplate
washer and blot plate on paper towels. Add 150 μL of 1×MSD Read
Buffer without surfactant (Meso Scale Discovery). Read emitted
luminescence light at 620 nm on Sector Imager 6000 reader (Meso Scale
Discovery). The ECL values were analyzed with the concentrations of the
test antibodies used in the assay, using a four-parameter nonlinear least
squares fit, to obtain the IC50 values of each competitor in the
assay.

Results and Discussion:

[0555] Fifteen unique phage antibodies derived from YW243.55 that bound
both human and murine PD-L1 were chosen and reformatted to full length
IgG1 antibodies for further evaluation. The light and heavy chain
variable region sequences of these antibodies are reported in FIGS. 11A
and B.

[0559] This example shows the specificity for the anti-PD-L1 antibody of
the invention for human, rhesus and mouse PD-L1. In addition, it shows
the affinity of the Ab for mouse and human PD-L1 expressed at the cell
membrane on 293-transfected cells.

[0560] Human and mouse PD-L1 were stably transfected into 293 cells. Cells
were harvested and plated at 150,000 cells per well in a 96-well plate
for binding studies.

[0561] Rhesus blood was obtained from Southwest Foundation for Biomedical
Research (San Antonio, Tex.). Blood was diluted with an equal volume of
PBS and overlayed on 96% Ficoll-Paque (GE Healthcare) for separation of
mononuclear cells. Mononuclear cells were lysed of red blood cells using
erythrocyte lysis buffer (Qiagen) and cultured overnight at
1.5×106 cells/ml with 5 ng/ml PMA plus 1 μM ionomycin in
6-well plates. Culture media was RPMI 1640 with 10% fetal bovine serum,
20 μM HEPES, and 1:100 dilutions of the following supplements from
Gibco: Gluta-MAX, sodium pyruvate, penicillin/streptomycin, and
non-essential amino acids. Cells were harvested the following day and
aliquoted to a 96-well plate for binding studies (approximately 120,000
cells per well).

[0562] The PD-L1 antibody YW243.55.570 or Herceptin® antibody control
were titrated starting at 10 μg/ml, in three-fold serial dilutions and
bound to cells in 50 μl volumes for 25 minutes on ice. Cells were
washed and then bound with anti-human IgG PE (Caltag) at 20 μg/ml for
25 minutes on ice. Rhesus cells were also co-stained with CD3 FITC and
CD4 APC (BD Biosciences) to distinguish CD4+ T cells.

[0563] All samples were run on a Beckman Dickinson FACSCalibur and Mean
Fluorescence Intensity of PD-L1 binding data as a function of anti-PD-L1
antibody concentration was analyzed using Tree Star, Inc. FlowJo®
software; EC50 values (Ab concentration associated with half-maximal
binding) were calculated using Kaleidagraph. In addition, equilibrium
binding studies were performed to define accurate affinities (Kds) for
YW24355570 binding to human and mouse PD-L1 expressed on 293 cells
(Example 3B). These values are summarized below in Table 3:

[0564] 293 cells transfected with human or mouse PD-L1 were cultured in
growth media, which consisted of RPMI 1640 media supplemented with 10%
fetal bovine serum (FBS), 2 mM L-glutamine, 1×
penicillin-streptomycin, at 37 degrees C. in 5% CO2. Cells were
washed with binding buffer (50:50 DMEM/F12 with 2% FBS and 50 mM Hepes,
pH 7.2) and were placed into 96-well plates at approximately 230,000
cells in 0.2 mL of binding buffer. The anti-PD-L1 antibody,
YW243.55.S70.hIgG, was iodinated using the Iodogen method. The
radiolabeled anti-PD-L1 antibodies were purified from free 125I-NA
by gel filtration using a NAP-5 column; the purified Ab had a specific
activity of 17.41 μCi/μg. Competition reaction mixtures of 50 μL
volume containing a fixed concentration of iodinated antibody and
decreasing concentrations of serially diluted unlabeled antibody were
placed into 96-well plates. 293 stable transfectant cell lines expressing
human PD-L1 and murine PD-L1 were cultured in growth media, which
consisted of 50:50 DMEM/F12 media supplemented with 10% fetal bovine
serum (FBS), 2 mM L-glutamine, 1× penicillin-streptomycin, at
37° C. in 5% CO2. Cells were washed with binding buffer
(50:50 DMEM/F12 with 2% FBS, 50 mM HEPES, pH 7.2, and 2 mM sodium azide)
and were added at an approximate density of 200,000 cells in 0.2 mL of
binding buffer to the 50 μL competition reaction mixtures. The final
concentration of the iodinated antibody in each competition reaction with
cells was ˜150 pM (˜120,000 cpms per 0.25 mL) and the final
concentration of the unlabeled antibody in the competition reaction with
cells varied, starting at 500 nM and then decreasing by 2 fold for 10
concentrations. Competition reactions with cells were incubated for 2
hours at room temperature. Competition reaction with cells for each
concentration of unlabeled antibody was assayed in triplicate. After the
2 hour incubation, the competition reactions were transferred to a
Millipore Multiscreen filter plate and washed 4× with binding
buffer to separate the free from bound iodinated antibody. The filters
were counted on a Wallac Wizard 1470 gamma counter (PerkinElmer Life and
Analytical Sciences Inc. Wellesley, Mass.). The binding data was
evaluated using NewLigand software (Genentech), which uses the fitting
algorithm of Munson and Robard to determine the binding affinity of the
antibody. Musson et al., Anal. Biochem. 107: 220-39 (1980).

[0565] The Kd values as determined by Scatchard analysis corroborates the
EC50 values of anti-PD-L1 antibody binding to human and mouse PD-L1 as
shown in Table 3.

Example 4

Selectivity and Affinity of Anti-PD-L1 Abs (IC50)

[0566] This example shows the binding selectivity and affinity (as
IC50) assay used to evaluate the full-length anti-PD-L1 antibodies
of the present invention for their ability to block binding of PD-L1 to
both PD-1 and B7.1.

[0568] Nunc Maxisorp 384 well plate was coated with 25 μL of 250 ng/mL
hPD-L1-Fc in PBS overnight. Wash wells three times with 0.05% Tween in
PBS (Wash Buffer) on a microplate washer and block wells with 0.5% BSA in
PBS. Add 12.5 μL of 2× concentration of antibodies to each well
in 0.05% Tween, 0.5% BSA in PBS (Assay Diluent) and followed by 12.5
μL of 250 ng/mL (2× concentration) of hB7.1-Fc-biotin in Assay
Diluent and incubate plate for one and half hour with agitation. Wash
wells six times with Wash Buffer and add 25 μL of Streptavidin-HRP
(1:40,000 in Assay Diluent, GE Healthcare). Incubate plate for 30 minutes
with agitation and wash wells six times with Wash Buffer. Add 25 μL of
TMB substrate (Kirkegaard and Perry Laboratories) for one hour and stop
reaction with 25 μL of 1 M Phosphoric Acid. Read absorbance at 450 nm
and analyze IC50 values as described under ECL cell-binding assay in
Example 1.

[0569] Formats 5, 6, 7:

[0570] For hPD-1-Fc-biotin binding to hPD-L1-Fc (Format 5), the format is
similar to the above assay except hPD-1-Fc-biotin was used instead of
hB7.1-Fc-biotin for binding. The TMB substrate reaction time was 17
minutes.

[0571] For mB7.1-Fc-biotin binding to mPD-L1-Fc (Format 6), the format is
similar to Format 5, except that mPD-L1-Fc was used to coat plate instead
of hPD-L1-Fc and mB7.1-Fc-biotin was used for binding instead of
hB7.1-Fc-biotin. The TMB substrate reaction time was 7 minutes.

[0572] For mPD-1-Fc-biotin binding to mPD-L1-Fc (Format 7), the format is
similar to the mouse ELISA mentioned above except mPD-1-Fc-biotin was
used for binding instead of mB7.1-Fc-biotin. The TMB substrate reaction
time was 5 minutes.

Results:

[0573] Assessment of the IC50 of the affinity-matured phage
anti-PD-L1 Antibody YW243.55.570 to block interactions between the
designated binding pairs is reported in Table 4. YW243.55570 was able to
block binding of human PD-L1 to hB7.1 Fc with a half-maximal inhibitory
concentration of 38 pM, a concentration relatively comparable to its
IC50 value for blocking the PD-L1/PD-1 interaction (42 pM). Biacore
studies measuring the capacity of YW243.55S70 to block both interactions
of PD-L1 with PD-1 and B7.1 were consistent with these ELISA results
(data not shown).

[0574] This example shows the effect of the anti-PD-L1 antibodies of the
invention upon activation of PMEL T cell receptor transgenic CD8.sup.+ T
cells, as measured by enhancement of γ-IFN production in response
to melanocyte peptide, gp100. In this procedure, CD8+ T cells are
obtained from PMEL TCR transgenic mice whose CD8+ T cells express a TCR
specific for the gp100 peptide. Following purification of the CD8+ T
cells, multiple rounds of stimulation are performed to generate and
expand the activated CD8+ T-cells, which will then in turn upregulate
PD-1 expression. In parallel, B16 melanoma cells are treated with
IFN-γ to upregulate their PD-L1 expression. Then, the cells are
co-cultured in the presence of anti-PD-L1 antibody, and the effect on
IFN-γ production is evaluated. B16 cells were chosen for the
tertiary stimulation because they endogenously express low levels of
gp100 peptide (as opposed to exogenous application of the peptide).
Moreover, as these cells also do not express PD-L2, B7.1 or B7.2, the
effect of additional signaling unrelated to PD-L1 (e.g. signaling through
CD28 or CTLA-4 or PD-L2 induced signaling through PD-1) is minimized.

[0575] PMEL Assay:

[0576] As shown in FIG. 3, anti-PD-L1 antibodies enhance both the
percentage of IFN-γ-producing PMEL CD8.sup.+ T cells and the
average levels of IFN-γ produced in response to the designated
amounts of gp100 peptide.

[0577] D.011.10 In Vitro Assay:

[0578] A similar assay utilizing Ova-specific TCR Tg CD4+ T cells shows
enhanced T cell proliferation in the presence of the anti-PD-L1 Ab
following prior stimulation with Ova peptide to induce expression of PD-1
(FIG. 4). In the final stimulation, irradiated A20 B cells that express
PD-L1 were used to present the designated concentrations of Ova peptide
to the D0.11.10 T cells. Notably, the contribution of the PD-1/PD-L1 axis
is more pronounced at lower degrees of antigen receptor stimulation,
levels that more closely reflect the physiologically relevant magnitude
of stimulation.

[0580] Spleen was isolated from a non-transgenic sex-matched mouse and
crushed into a single cell suspension and red blood cell lysed. Cells
were pulsed with 0.1 μg/ml of gp100-peptide for two hours at
37° C. and washed.

[0582] PMEL cultures were spun down and the media was aspirated using a
multi-channel pipet. Fresh media was added and mixed to wash the cells,
followed by another spin. Majority of the media was removed and
antibodies (Herceptin®, YW243.55.570, or none) were added for a final
concentration of 10 μg/ml. Conditions were set up in duplicate wells
such that the average IFN-γ production could be assessed at the
endpoint.

[0584] One day prior to third stimulation on day 6, B16 melanoma cells
were incubated with 20 ng/ml of mouse IFN-γ (R&D Systems) overnight
to upregulate their PD-L1 expression.

[0585] On day 7, PMEL cultures were spun down and the media was aspirated
using a multi-channel pipet. Fresh media was added and mixed, followed by
another spin. Majority of the media was removed and antibodies were added
for a final concentration of 10 μg/ml.

[0586] After overnight stimulation with IFN-γ, B16 cells were washed
and split into three groups for a two hour incubation with either no
gp100, gp100 at 1 ng/ml (gp100 low), and gp100 at 10 ng/ml (gp100 high).
Cells were washed and then added to the washed PMEL+Ab cultures at 40,000
cells per well and incubated together overnight.

Day 8 IFN-γ Intracellular Staining

[0587] Golgi-Plug (BD Biosciences) was added for the last 5 hours of
culture as per manufacturer's instructions. IFN-γ intracellular
staining was done using BD Biosciences Cytofix/Cytoperm
Fixation/Permeabilization Solution kit as per manufacturer's instructions
and all staining antibodies were also from BD Biosciences. Cells were
surface stained with CD8a PE and Thy1.1 FITC and intracellular stained
with IFN-γ APC at saturating concentrations.

[0588] All samples were run on a Beckman Dickinson FACSCalibur and data
was analyzed using Tree Star, Inc. FLOWJO® software.

D011.10 In Vitro Assay

[0589] Spleen and mesenteric lymph nodes from D011.10 transgenic mice were
harvested, crushed into single cell suspensions, and lysed of red blood
cells. Cells were cultured for 72 hours at a density of 1×106
cells per ml in 6 well plates with Ova peptide at 0.3 μM. Culture
media was RPMI 1640+10% fetal bovine serum+20 μM HEPES, and 1:100
dilutions of the following supplements from Gibco: Gluta-MAX, sodium
pyruvate, penicillin/streptomycin, and non-essential amino acids.

[0590] After the primary stimulation, cells were harvested and purified
for CD4.sup.+ T cells using a mouse CD4 T cell purification kit as per
manufacturer's instructions (Miltenyi Biotec). Purified CD4.sup.+ T cells
were then rested overnight.

[0591] The next day, cells were harvested, washed, and co-cultured with
irradiated (10,000 rads) A20 cells. Co-culture was set up in 96-well U
bottom plates in triplicate wells, with 50,000 CD4.sup.+ T cells to
40,000 A20 cells with titrated Ova peptide and antibody at a final
concentration of 20 μg/ml. After 48 hours, cultures were pulsed
overnight with 1 μCi/well of 3H-thymidine and frozen the next day.
Plates were later thawed, harvested on a cell harvester, and read on a
beta-counter.

[0592]FIG. 5 demonstrates the ability of anti-PD-L1 (e.g., YW243.55.S1)
to enhance proliferation of human CD8 T cells in response to cells from
an MHC-mismatched donor. Responding CD8+ T cells were enriched from whole
blood of Donor A by first using CD8+ T cell RosetteSep® (StemCell
Technologies) as per manufacturer's instructions. Cells were then diluted
by an equal volume of phosphate buffered saline (PBS) and separated by
gradient centrifugation by overlaying on Ficoll-Paque Plus (GE
Healthcare). After separation, cells were stained with CD8 APC (BD
Biosciences) and found to be 78% CD8+ T cells. Cells were fluorescently
labeled with 2.5 μM CFSE tracer dye (Molecular Probes).

[0593] To serve as allogeneic antigen presenting cells (APCs), mononuclear
cells were first isolated from whole blood from Donor B and then depleted
of CD3+ T cells. Blood was diluted with an equal volume of PBS and
mononuclear cells were isolated after gradient centrifugation over
Ficoll. Cells were stained with CD3 FITC (BD Biosciences), washed, and
then incubated with anti-FITC microbeads (Miltenyi Biotec). CD3 FITC
positive cells were then depleted on the AutoMACS cell separator
(Miltenyi Biotec). Cells were then irradiated 2500 rads in a cesium
irradiator.

[0594] Cells were co-cultured in a 96-well flat-bottom plate with 150,000
CD8+ T cells and 150,000 APCs for 5 days with antibodies at 10 μg/ml.
Culture media was RPMI 1640+10% fetal bovine serum+20 μM HEPES, and
1:100 dilutions of the following supplements from Gibco: Gluta-MAX,
sodium pyruvate, penicillin/streptomycin, and non-essential amino acids.

[0595] On day 5, cells were harvested, washed and stained with CD8-biotin
followed by streptavidin-PerCp (BD Biosciences). Samples were run on a
Beckman Dickinson FACSCalibur and data was analyzed using Tree Star, Inc.
FlowJo software.

[0596] An approximately 45% enhancement in proliferation of CD8 T cells
responding to cells from an MHC-mismatched donor was observed in the
presence of the anti-PD-L1.

Example 7

Effects of PD-L1 Blockade on LCMV In Vivo Model

[0597] T cells under conditions of chronic stimulation have been shown to
upregulate and sustain expression of the inhibitory receptor PD-1.
Ligation of PD-1 by either of its two ligands PD-L1 and PD-L2 contributes
to the refractory state of the chronically activated T cell, attenuating
its response to its cognate antigen. In mice persistently-infected with
lymphocytic choriomeningitis virus (LCMV), blockade of PD-1 or its ligand
PD-L1 is sufficient to revitalize chronically refractory T cells,
enhancing the magnitude and functional quality of the anti-viral T cell
response. Similarly, humans chronically infected with HIV or HCV exhibit
T cells refractory to simulation whose activity can be enhanced in vitro
by blockade of PD-1 or PD-L1. Therefore, activity of PD-L1 blockade in
the LCMV model suggests therapeutic potential for enhancing anti-viral
and anti-tumor immunity.

[0598] For the LCMV in vivo experiments in the mouse, we have reformatted
the humanized anti-PD-L1 antibody (YW243.55570), by cloning the
phage-derived heavy and light chain variable region sequences upstream of
mouse IgG2a heavy chain and mouse kappa light chain constant domains. To
prevent antibody-mediated cytotoxicity of PD-L1 expressing cells, by
inhibiting Fcγ receptor binding, positions 265 (aspartic acid) and
297 (asparagine) were changed to alanine (DANA). Shields, R L et al J.
Biol Chem 2001 276 (9): 6591-6604. To test the ability of the anti-PD-L1
antibody to enhance anti-viral immunity in a chronic infection, mice were
infected at Day 0 with 2×106 plaque forming units (pfu) of
Clone 13 LCMV or the Armstrong strain of LCMV as a reference control. The
schematic of the experimental design appears in FIG. 6. Infection with
Clone 13 results in a chronic infection, characterized by T cells that
expand but are unable to effectively clear the virus, while Armstrong
LCMV is cleared within 8-10 days of infection. On day 14, mice began
treatment with either anti-PD-L1 or control mIgG delivered at 10 mg/kg
doses 3×/week. At Days 21 and 28, analysis of CD8 T cell function
and viral titers in blood and tissues were performed.

[0599] Consistent with published data of Barber et. al, Nature 439:682-7
(2006), this example shows the ability of the anti-PD-L1 Ab to enhance
the cytotoxic lymphocyte response to LCMV following a 2 week treatment
regimen in a chronic LCMV infection. FIG. 7A shows the % of CD8 T cells
that express CD107a on their cell surface in response to gp33
LCMV-specific peptide. Plasma membrane expression of CD107a, normally
expressed intracellularly, accompanies the degranulation process and
therefore serves as a surrogate marker for degranulation. Relative to the
response of cells from the acute Armstrong LCMV infection, cells from
animals infected with the chronic strain, clone 13, are impaired in
degraulation (control Ig group), while PD-L1 blockade was able to restore
CD8+degraulation to levels comparable to those observed in the Armstrong
infection. Similarly, 7B demonstrates the increased % of
IFN-γ-producing CD8 T cells in response to LCMV gp33 in the
anti-PD-L1-treated group relative to control Ig.

[0600] Next, the impact of the anti-PD-L1 Ab on reducing or eradicating
LCMV virus in blood and tissues was tested. In FIG. 8A, the graphs show
log virus titers in the indicated tissue of control Ig and PD-L1 treated
animals at day 21 and 28 after infection with Clone 13 LCMV. Antibody
treatment was initiated at Day 14 post-infection. Blockade of PD-L1
resulted in highly significant reduction in viral titers in blood, liver,
brain, lung, and kidney. Impressively, in 3 of 5 mice, α-PD-L1 Ab
reduced blood LCMV titers to levels below detection
(<1×10-5). In a subsequent experiment of comparable design,
virus eradication in blood and liver was observed in 5/5 mice treated for
2 weeks with anti-PD-L1 at doses of either 10 mg/kg or 2 mg/kg
3×/week (data not shown). The lower graph shows the kinetics of
reduction of viral titers in the blood and demonstrates an average
reduction of 96.8% in the anti-PD-L1 treated group at Day 28 relative to
control. These data support the importance of the PD-1/PD-L1 pathway in
inhibiting T cell responses in chronic infections and are consistent with
effects of in vitro PD-L1 blockade on T cells obtained from humans with
chronic infections such as Hepatitis C and HIV.

[0603] MC57 fibrosarcoma cells are infected with 10-fold serial dilutions
of LCMV-containing blood or tissue homogenate in complete IMDM. The
reaction is then incubated for 2-6 hours in at 37° C. in a tissue
culture incubator, then overlayed with DMEM containing 1%
methylcellulose. This is followed by incubation for 3-5 days, then the
methylcellulose layer is removed by aspiration. The cells are fixed with
PBS/4% paraformaldehyde, then permeabilized with 0.5% Triton-x for 20
minutes, washed in PBS, then blocked in 10% FCS for 1 hour with mild
rocking. Staining for LCMV is done with VL4 antibody (1 hour), washed
2×, then developed with anti-rat HRP (1:400) in blocking buffer.
This followed by washing 3×, then adding o-phenylenediamine
substrate (SIGMA P8806-50TAB 3 mg/tablet) to wells to develop.

Example 8

PD-L1 Blockade in Cancer

[0604] It is now apparent that many tumors exploit expression of PD-1
ligands as a means to attenuate anti-tumor T cells responses. Several
human cancers have been characterized to express elevated levels of PD-L1
on both tumors and tumor-infiltrating leukocytes and this elevated PD-L1
expression is often associated with a worse prognosis. Mouse tumor models
demonstrate similar increases in PD-L1 expression within tumors and
demonstrate a role for the PD-1/PD-L1 pathway in inhibiting tumor
immunity.

[0605] Here we present an experiment demonstrating the impact of blocking
PD-L1 on orthotopic tumor growth of MC38.Ova murine colorectal carcinoma
cells in syngeneic C57B6 mice (FIG. 9A). These cells express ovalbumin
via retroviral transduction and express PD-L1, but not PD-L2 on their
cell surface as assessed by Flow Cytometry (histogram--FIG. 10A). Mice
were inoculated subcutaneously with 0.5 million MC38.Ova cells on Day 0.
On Day 1 or on Day 14 mice (when tumors had reached an average size of
250 mm3) 10 mice/group were treated with 10 mg/kg anti-PD-L1
(YW243.55S70-mouse IgG2a-DANA), control Ig, or blocking anti-CTLA4 Ab,
(UC10-4F10-11) 3×/week for the duration of the study. Blockade of
PD-L1 either early or in late intervention is highly effective as a
single agent therapy at preventing tumor growth. In contrast, blockade of
CTLA4, another inhibitory molecule expressed on T cells showed no
evidence of inhibiting tumor growth. These results demonstrate the unique
role of the PD-1/PD-L1 axis over CTLA4/B7 in suppression of the
anti-tumor immune response and support the potential for the treatment of
human cancers with antibodies that block the PD-L1 interaction with PD-1
and B7.1.

MC38.Ova Syngeneic Tumor Model: Methods.

[0606] On Day 0, 70 animals were inoculated subcutaneously with 0.5
million MC38.Ova cell in 100 microliters of HBSS+matrigel. Beginning on
D1, 20 mice were recruited into one of 2 treatment groups (see below:
group 1 or group 2). The remaining 40 mice were allowed to grow tumors
until Day 14. Of these 40, 30 mice with similar-sized tumors were
recruited into one of 3 treatment groups (Groups 3-5). The tumors were
measured and the mice weighed 2×/week. Mice not recruited into
below treatment groups, due to dissimilar tumor volume were euthanized:

Combinations of Anti-PD-L1 with Other Agents to Provide for Anti-Tumor
Effect or Immune-Enhancing Therapy

MC38.Ova Model

[0607] On Day 0, 150 animals are inoculated subcutaneously with 0.5
million MC38.Ova cell in 100 microliters of HBSS+matrigel. Mice are
allowed to grow tumors. Mice are weighed and measured 2×/week until
Day 11 (when the tumor volume is between 100-200 mm3). On Day 11,
following tumor measurement, mice are recruited into 1 of the 12
treatment groups below. Mice not recruited into below treatment groups,
due to dissimilar tumor volume are euthanized. Gemcitabine (Group 4)
treatment starts on day 12, while treatment for the remaining antibody
groups starts on day 14. All volumes are 100 μl in inert vehicle, with
additional details as reported below:

[0608] Day 12: Mice from group 1 are bled (100
microliters) retro-orbitally under anaesthesia for CBC analysis.

[0609]
Day 14 and Day 22: Mice from group 4 are bled (100 microliters)
retro-orbitally under anaesthesia for CBC analysis.

[0610] Day 19: All
mice, except group 4, are bled (100 microliters) retro-orbitally under
anaesthesia for CBC analysis.

[0611] Day 26: All mice, except group 4,
are bled (100 microliters) retro-orbitally under anaesthesia for PK
analysis.

[0612] Tumors are measured and mice weighed 2×/week. Animals
exhibiting weight loss of >15% will be weighed daily and euthanized if
they lose >20% body weight. Mice will be euthanized when tumor volumes
exceed 3,000 mm3, or after 3 months if tumors do not form.

[0613] This study shows (FIG. 10) that PD-L1 blockade was more effective
than a-VEGF and an inductive regimen of gemcitabine alone.

[0615] The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is
employed as the expression vector. Optionally, DNA encoding the light
and/or heavy chain of the antibody is ligated into pRK5 with selected
restriction enzymes to allow insertion such DNA using ligation methods
such as described in Sambrook et al., supra.

[0616] In one embodiment, the selected host cells may be 293 cells. Human
293 cells (ATCC CCL 1573) are grown to confluence in tissue culture
plates in medium such as DMEM supplemented with fetal calf serum and
optionally, nutrient components and/or antibiotics. About 10 μg if DNA
encoding the pRK5-antibody is mixed with about 1 μg DNA encoding the
VA RNA gene [Thimmappaya et al., Cell, 31:543 (1982)] and dissolved in
500 μL of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl2. To this
mixture is added, dropwise, 500 μL of 50 mM HEPES (pH 7.35), 280 mM
NaCl, 1.5 mM NaPO4, and a precipitate is allowed to form for 10
minutes at 25° C. The precipitate is suspended and added to the
293 cells and allowed to settle for about four hours at 37° C. The
culture medium is aspirated off and 2 ml of 20% glycerol in PBS is added
for 30 seconds. The 293 cells are then washed with serum free medium,
fresh medium is added and the cells are incubated for about 5 days.

[0617] Approximately 24 hours after the transfections, the culture medium
is removed and replaced with culture medium (alone) or culture medium
containing 200 μCi/ml 35S-cysteine and 200 μCi/ml
35S-methionine. After a 12 hour incubation, the conditioned medium
is collected, concentrated on a spin filter, and loaded onto a 15% SDS
gel. The processed gel may be dried and exposed to film for a selected
period of time to reveal the presence of the antibody. The cultures
containing transfected cells may undergo further incubation (in serum
free medium) and the medium is tested in selected bioassays.

[0618] In an alternative technique, the antibody may be introduced into
293 cells transiently using the dextran sulfate method described by
Somparyrac et al., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells are
grown to maximal density in a spinner flask and 700 μg DNA encoding
the pRK5-antibody is added. The cells are first concentrated from the
spinner flask by centrifugation and washed with PBS. The DNA-dextran
precipitate is incubated on the cell pellet for four hours. The cells are
treated with 20% glycerol for 90 seconds, washed with tissue culture
medium, and re-introduced into the spinner flask containing tissue
culture medium, 5 μg/ml bovine insulin and 0.1 μg/ml bovine
transferrin. After about four days, the conditioned media is centrifuged
and filtered to remove cells and debris. The sample containing the
expressed antibody can then be concentrated and purified by any selected
method, such as dialysis and/or column chromatography.

[0619] In another embodiment, the antibody can be expressed in CHO cells.
The DNA encoding the antibody ligated into pRK5 can be transfected into
CHO cells using known reagents such as CaPO4 or DEAE-dextran. As
described above, the cell cultures can be incubated, and the medium
replaced with culture medium (alone) or medium containing a radiolabel
such as 35S-methionine. After determining the presence of the
antibody, the culture medium may be replaced with serum free medium.
Preferably, the cultures are incubated for about 6 days, and then the
conditioned medium is harvested. The medium containing the expressed
antibody can then be concentrated and purified by any selected method.

[0620] Epitope-tagged variants of the antibody may also be expressed in
host CHO cells. The DNA encoding the antibody ligated into pRK5 may be
subcloned out of the pRK5 vector. The subclone insert can undergo PCR to
fuse in frame with a selected epitope tag such as a poly-his tag into a
Baculovirus expression vector. The poly-his tagged DNA encoding the
antibody insert can then be subcloned into a SV40 driven vector
containing a selection marker such as DHFR for selection of stable
clones. Finally, the CHO cells can be transfected (as described above)
with the SV40 driven vector. Labeling may be performed, as described
above, to verify expression. The culture medium containing the expressed
poly-His tagged antibody can then be concentrated and purified by any
selected method, such as by Ni2+-chelate affinity chromatography.

[0621] The antibody may also be expressed in CHO and/or COS cells by a
transient expression procedure or in CHO cells by another stable
expression procedure.

[0622] Stable expression in CHO cells is performed using the following
procedure. The proteins are expressed as an IgG construct
(immunoadhesin), in which the coding sequences for the soluble forms
(e.g. extracellular domains) of the respective proteins are fused to an
IgG1 constant region sequence containing the hinge, CH2 and CH2 domains
and/or is a poly-His tagged form.

[0623] Following PCR amplification, the respective DNAs are subcloned in a
CHO expression vector using standard techniques as described in Ausubel
et al., Current Protocols of Molecular Biology, Unit 3.16, John Wiley and
Sons (1997). CHO expression vectors are constructed to have compatible
restriction sites 5' and 3' of the DNA of interest to allow the
convenient shuttling of cDNA's. The vector used expression in CHO cells
is as described in Lucas et al., Nucl. Acids Res. 24:9 (1774-1779 (1996),
and uses the SV40 early promoter/enhancer to drive expression of the cDNA
of interest and dihydrofolate reductase (DHFR). DHFR expression permits
selection for stable maintenance of the plasmid following transfection.

[0624] Twelve micrograms of the desired plasmid DNA is introduced into
approximately 10 million CHO cells using commercially available
transfection reagents SUPERFECT® (Quiagen), DOSPER® or
FUGENE® (Boehringer Mannheim). The cells are grown as described in
Lucas et al., supra. Approximately 3×10-7 cells are frozen in
an ampule for further growth and production as described below.

[0625] The ampules containing the plasmid DNA are thawed by placement into
water bath and mixed by vortexing. The contents are pipetted into a
centrifuge tube containing 10 mLs of media and centrifuged at 1000 rpm
for 5 minutes. The supernatant is aspirated and the cells are resuspended
in 10 mL of selective media (0.2 μm filtered PS20 with 5% 0.2 μm
diafiltered fetal bovine serum). The cells are then aliquoted into a 100
mL spinner containing 90 mL of selective media. After 1-2 days, the cells
are transferred into a 250 mL spinner filled with 150 mL selective growth
medium and incubated at 37° C. After another 2-3 days, 250 mL, 500
mL and 2000 mL spinners are seeded with 3×105 cells/mL. The
cell media is exchanged with fresh media by centrifugation and
resuspension in production medium. Although any suitable CHO media may be
employed, a production medium described in U.S. Pat. No. 5,122,469,
issued Jun. 16, 1992 may actually be used. A 3 L production spinner is
seeded at 1.2×106 cells/mL. On day 0, the cell number and pH
is determined. On day 1, the spinner is sampled and sparging with
filtered air is commenced. On day 2, the spinner is sampled, the
temperature shifted to 33° C., and 30 mL of 500 g/L glucose and
0.6 mL of 10% antifoam (e.g., 35% polydimethylsiloxane emulsion, Dow
Corning 365 Medical Grade Emulsion) taken. Throughout the production, the
pH is adjusted as necessary to keep it at around 7.2. After 10 days, or
until the viability dropped below 70%, the cell culture is harvested by
centrifugation and filtering through a 0.22 μm filter. The filtrate
was either stored at 4° C. or immediately loaded onto columns for
purification.

[0626] For the poly-His tagged constructs, the proteins are purified using
a Ni-NTA column (Qiagen). Before purification, imidazole is added to the
conditioned media to a concentration of 5 mM. The conditioned media is
pumped onto a 6 ml Ni-NTA column equilibrated at 4° C., in 20 mM
Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow
rate of 4-5 ml/min. After loading, the column is washed with additional
equilibration buffer and the protein eluted with equilibration buffer
containing 0.25 M imidazole. The highly purified protein is subsequently
desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4%
mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and
stored at -80° C.

[0627] Immunoadhesin (Fc-containing) constructs are purified from the
conditioned media as follows. The conditioned medium is pumped onto a 5
ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na
phosphate buffer, pH 6.8. After loading, the column is washed extensively
with equilibration buffer before elution with 100 mM citric acid, pH 3.5.
The eluted protein is immediately neutralized by collecting 1 ml
fractions into tubes containing 275 μL of 1 M Tris buffer, pH 9. The
highly purified protein is subsequently desalted into storage buffer as
described above for the poly-His tagged proteins. The homogeneity is
assessed by SDS polyacrylamide gels and by N-terminal amino acid
sequencing by Edman degradation.

Example 11

Expression of Anti-PD-L1 Antibody in E. coli

[0628] This example illustrates preparation of an unglycosylated form of
anti-PD-L1 antibody by recombinant expression in E. coli.

[0629] The DNA sequence encoding the anti-PD-L1 antibody is initially
amplified using selected PCR primers. The primers should contain
restriction enzyme sites which correspond to the restriction enzyme sites
on the selected expression vector. A variety of expression vectors may be
employed. An example of a suitable vector is pBR322 (derived from E.
coli; see Bolivar et al., Gene, 2:95 (1977)) which contains genes for
ampicillin and tetracycline resistance. The vector is digested with
restriction enzyme and dephosphorylated. The PCR amplified sequences are
then ligated into the vector. The vector will preferably include
sequences which encode for an antibiotic resistance gene, a trp promoter,
a polyhis leader (including the first six STII codons, polyhis sequence,
and enterokinase cleavage site), the NPOR coding region, lambda
transcriptional terminator, and an argU gene.

[0630] The ligation mixture is then used to transform a selected E. coli
strain using the methods described in Sambrook et al., supra.
Transformants are identified by their ability to grow on LB plates and
antibiotic resistant colonies are then selected. Plasmid DNA can be
isolated and confirmed by restriction analysis and DNA sequencing.

[0631] Selected clones can be grown overnight in liquid culture medium
such as LB broth supplemented with antibiotics. The overnight culture may
subsequently be used to inoculate a larger scale culture. The cells are
then grown to a desired optical density, during which the expression
promoter is turned on.

[0632] After culturing the cells for several more hours, the cells can be
harvested by centrifugation. The cell pellet obtained by the
centrifugation can be solubilized using various agents known in the art,
and the solubilized antibody can then be purified using a metal chelating
column under conditions that allow tight binding of the antibody.

[0633] Anti-PD-L1 antibody may also be expressed in E. coli in a poly-His
tagged form, using the following procedure. The DNA encoding antibody is
initially amplified using selected PCR primers. The primers contain
restriction enzyme sites which correspond to the restriction enzyme sites
on the selected expression vector, and other useful sequences providing
for efficient and reliable translation initiation, rapid purification on
a metal chelation column, and proteolytic removal with enterokinase. The
PCR-amplified, poly-His tagged sequences are then ligated into an
expression vector, which is used to transform an E. coli host based on
strain 52 (W3110 fuhA(tonA) lon galE rpoHts(htpRts) clpP(lacIq).
Transformants are first grown in LB containing 50 mg/ml carbenicillin at
30° C. with shaking until an O.D.600 of 3-5 is reached. Cultures
are then diluted 50-100 fold into CRAP media (prepared by mixing 3.57 g
(NH4)2SO4, 0.71 g sodium citrate.2H2O, 1.07 g KCl,
5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500 mL water,
as well as 110 mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgSO4)
and grown for approximately 20-30 hours at 30° C. with shaking.
Samples are removed to verify expression by SDS-PAGE analysis, and the
bulk culture is centrifuged to pellet the cells. Cell pellets are frozen
until purification and refolding.

[0634] E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) is
resuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8
buffer. Solid sodium sulfite and sodium tetrathionate is added to make
final concentrations of 0.1M and 0.02 M, respectively, and the solution
is stirred overnight at 4° C. This step results in a denatured
protein with all cysteine residues blocked by sulfitolization. The
solution is centrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30
min. The supernatant is diluted with 3-5 volumes of metal chelate column
buffer (6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22
micron filters to clarify. Depending on the condition, the clarified
extract is loaded onto a 5 ml Qiagen Ni-NTA metal chelate column
equilibrated in the metal chelate column buffer. The column is washed
with additional buffer containing 50 mM imidazole (Calbiochem, Utrol
grade), pH 7.4. The protein is eluted with buffer containing 250 mM
imidazole. Fractions containing the desired protein were pooled and
stored at 4° C. Protein concentration is estimated by its
absorbance at 280 nm using the calculated extinction coefficient based on
its amino acid sequence.

[0635] The proteins are refolded by diluting sample slowly into freshly
prepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3 M NaCl,
2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. Refolding volumes
are chosen so that the final protein concentration is between 50 to 100
micrograms/ml. The refolding solution is stirred gently at 4° C.
for 12-36 hours. The refolding reaction is quenched by the addition of
TFA to a final concentration of 0.4% (pH of approximately 3). Before
further purification of the protein, the solution is filtered through a
0.22 micron filter and acetonitrile is added to 2-10% final
concentration. The refolded protein is chromatographed on a Poros R1/H
reversed phase column using a mobile buffer of 0.1% TFA with elution with
a gradient of acetonitrile from 10 to 80%. Aliquots of fractions with
A280 absorbance are analyzed on SDS polyacrylamide gels and fractions
containing homogeneous refolded protein are pooled. Generally, the
properly refolded species of most proteins are eluted at the lowest
concentrations of acetonitrile since those species are the most compact
with their hydrophobic interiors shielded from interaction with the
reversed phase resin. Aggregated species are usually eluted at higher
acetonitrile concentrations. In addition to resolving misfolded forms of
proteins from the desired form, the reversed phase step also removes
endotoxin from the samples.

[0636] Fractions containing the desired folded anti-PD-L1 antibodies are
pooled and the acetonitrile removed using a gentle stream of nitrogen
directed at the solution. Proteins are formulated into 20 mM Hepes, pH
6.8 with 0.14 M sodium chloride and 4% mannitol by dialysis or by gel
filtration using G25 Superfine (Pharmacia) resins equilibrated in the
formulation buffer and sterile filtered.